IMPROVED CELL-PERMEABLE CRE (ICP-CRE) RECOMBINANT PROTEIN AND USE THEREOF

Description

[Technical Field]

[0001] The present invention relates to improved cell-permeable (iCP) Cre recombinant protein and use thereof. The recombinant protein provides improved cell-/tissue-permeability, great intranuclear delivery and biological activity as a site-specific recombinase for researching the function of target gene.

[Background Art]

[0002] Epigenetics, that over or above genetics, refers to hereditary changes in genome expression that do not involve alteration of DNA sequences. Epigenetics is a study for physiological phenotypic trait variations that are caused by external or environmental factors that switch genes on and off. Hence, improvement of epigenetic research relies on a wide range of gene editing technology.

[0003] The gene editing technology is the most powerful tool to insert, replace, and delete targeted DNA from genome. DNA sequence-specific recombination has been widely used for the gene editing technology to regulate genetic modifications, such as conditional gene expression, conditional mutagenesis, gene replacement and chromosome engineering in mammalian. There are several engineered nucleases being used: Transcription Activator-Like Effector Nucleases (TALENs), CRISPER/Cas9 system, Sleeping Beauty, PiggyBac, Cre/LoxP system, and Flp/Frt systems.

[0004] Cre-mediated recombination has been widely used to manipulate the genomes of mammalian and non-mammalian organism. The Cre (Cyclization Recombinase) derived from bacteriophage P1 recognizes LoxP sites, which is comprised of 34 base pair sequences. A segment of DNA, which is flanked by the LoxP sites, is deleted by the Cre mediated recombination. The manipulation of the mouse genome has been enabled to access by the Cre/LoxP system. A common application of the Cre/LoxP system is to create conditional knockouts in mice. LoxP sites are introduced into the germ line. The mice are mated with a strain that expresses Cre in a tissue or developmentally restricted manner causing recombination of floxed gene to occur only in specific tissues or at specific times in development.

[0005] The site-specific recombination has also been used to manipulate mammalian chromosome, to insert exogenous DNA at specific sites in the genome, to promote activity of specific genes, and to suppress activity of specific genes. However, spatial- and temporal-controlled gene activation or deletion is often hampered by difficulties expressing the recombinase in the cells at the desired time and place. Plasmid and viral expression vectors are frequently used; however, the efficiency of DNA-mediated gene transfer is low. In addition, the early gene disruptions during embryogenesis by tissue-specific Cre expression in Cre Knock-in mice may cause abnormal development that leads to embryonic lethality. This fetal problem results in the limitation to study in terminally differentiated cells.

[References]

[0006] 

1. Fischer PM., Cellular uptake mechanisms and potential therapeutic utility of peptidic cell delivery vectors: progress 2001-2006, Med Res Rev. 2007;27:755-95.

2. Heitz F, Morris MC, Divita G., Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Br J Pharmacol. 2009;157:195-206.

3. Lapenna S, Giordano A., Cell cycle kinases as therapeutic targets for cancer, Nat Rev Drug Discov. 2009;8:547-66.

4. Lim J, Kim J, Duong T, Lee G, Kim J, Yoon J. et al., Antitumor activity of cell-permeable p18(INK4c) with enhanced membrane and tissue penetration, Mol Ther. 2012;20:1540-9.

5. Jo D, Liu D, Yao S, Collins RD, Hawiger J., Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis, Nat Med. 2005;11:892-8.

6. Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J. et al., Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase, Nat Biotechnol. 2001;19:929-33.

7. Liu D, Li C, Chen Y, Burnett C, Liu XY, Downs S. et al., Nuclear import of proinflammatory transcription factors is required for massive liver apoptosis induced by bacterial lipopolysaccharide, J Biol Chem. 2004;279:48434-42.

8. Liu D, Liu XY, Robinson D, Burnett C, Jackson C, Seele L. et al., Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor, J Biol Chem. 2004;279:19239-46.

9. Liu D, Zienkiewicz J, DiGiandomenico A, Hawiger J., Suppression of acute lung inflammation by intracellular peptide delivery of a nuclear import inhibitor, Mol Ther. 2009;17:796-802.

10. Moore DJ, Zienkiewicz J, Kendall PL, Liu D, Liu X, Veach RA. et al., In vivo islet protection by a nuclear import inhibitor in a mouse model of type 1 diabetes, PLoS One. 2010;5:e13235.

11. Lim J, Jang G, Kang S, Lee G, Nga do TT, Phuong do TL. et al., Cell-.permeable NM23 blocks the maintenance and progression of established pulmonary metastasis, Cancer Res. 2011;71:7216-25.

12. Duong T, Kim J, Ruley HE, Jo D., Cell-permeable parkin proteins suppress Parkinson disease-associated phenotypes in cultured cells and animals, PLoS One. 2014;9:e102517.

13. Lim J, Duong T, Do N, Do P, Kim J, Kim H. et al., Antitumor activity of cell-permeable RUNX3 protein in gastric cancer cells. Clin Cancer Res. 2013;19:680-90.

14. Lim J, Duong T, Lee G, Seong BL, El-Rifai W, Ruley HE et al. The effect of intracellular protein delivery on the anti-tumor activity of recombinant human endostatin, Biomaterials. 2013;34:6261-71.

15. Lim J, Kim J, Kang J, Jo D., Partial somatic to stem cell transformations induced by cell-permeable reprogramming factors, Scientific Reports. 2014;4:4361.

16. Sauer B. Inducible gene targeting in mice using the Cre/lox system. Methods 1998;14(4):381-92.

17. Betz UA, Vosshenrich CA, Rajewsky K, Muller W. Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination. Current Biology: CB 1996;6(10): 1307-16.

18. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science 1995;269(5229): 1427-9.

19. Lakso M, Sauer B, Mosinger B, Jr., Lee EJ, Manning RW, Yu SH, et al. Targeted oncogene activation by site-specific recombination in transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 1992;89(14):6232-6.

20. Smith AJ, De Sousa MA, Kwabi-Addo B, Heppell-Parton A, Impey H, Rabbitts P. A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature genetics 1995;9(4):376-85.

21. Chen CM, Behringer RR. CREating breakthroughs. Nature Biotechnology 2001;19(10):921-2.

22. Kolb AF, Siddell SG. Genomic targeting with an MBP-Cre fusion protein. Gene 1996; 183(1-2):53-60.

23. Baubonis W, Sauer B. Genomic targeting with purified Cre recombinase. Nucleic Acids Research 1993;21(9):2025-9.

24. Veach RA, Liu D, Yao S, Chen Y, Liu XY, Downs S et al.,Receptor/transporter-independent targeting of functional peptides across the plasma membrane. J Biol Chem 2004;279(12):11425-31.

25. Lim J, Kim J, Duong T, Lee G, Kim J, Yoon J et al., Antitumor activity of cell-permeable p18(INK4c) with enhanced membrane and tissue penetration. Molecular Therapy : the Journal of the American Society of Gene Therapy 2012;20(8): 1540-9.

26. Jo D, Nashabi A, Doxsee C, Lin Q, Unutmaz D, Chen J et al., Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nature Biotechnology 2001;19(10):929-33.

27. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J., Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. The Journal of Biological Chemistry 1995;270(24): 14255-8.

28. Jo D, Lin Q, Nashabi A, Mays DJ, Unutmaz D, Pietenpol JA et al., Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins. Journal of Cellular Biochemistry 2003;89(4):674-87.

[0007] WO 0220737 describes fusion proteins comprising Cre and a membrane translocation sequence from FGF-4. Dodecapeptide AAVLLPVLLAAP as described in Fig. 1 conferred an improved cell permeability to Cre recombinase and was used for Cre-mediated recombination in a transgenic mouse line.

[Disclosure]

[Technical Problem]

[0008] A macromolecule, such as Cre (Cyclization Recombinase) protein, cannot be translocated across the cell membrane; furthermore, it cannot be transported into the nucleus of the cell. Therefore, there was a need to develop macromolecule intracellular transduction technology (MITT), which enables the translocation of macromolecules into the cell/tissues.

[0009] In the previous studies, MITT-based hydrophobic CPPs named membrane translocating sequence (MTS) and membrane translocating motif (MTM), derived from the hydrophobic signal peptide of fibroblast growth factor 4 (FGF4) have been reported and used to deliver biologically active peptides and proteins, such as Cre protein, systemically in animals.

[0010] However, they could not effectively deliver Cre protein in vivo, and their delivery efficiency in vitro was also insufficient due to protein aggregation, low solubility/yield and poor cell-/tissue-permeability.

[Technical Solution]

[0011] To overcome the limitations and improve CPPs that provide cell-permeability of macromolecules in vitro and in vivo, theoretical critical factors (CFs) to improve the intracellular delivery potential of the CPPs are identified and verified according to one embodiment of the present invention. Based on the CFs determined, hydrophobic CPP sequences are newly created, quantitatively evaluated for cell-permeability and mutually compared to reference CPP sequences in their intracellular delivery potential in live cells. One embodiment of the present invention, newly developed hydrophobic CPPs are presented. The novel peptide sequences termed 'advanced macromolecule transduction domains' (aMTDs) could systematically deliver the aMTD-fused recombinant proteins to live cells and animal tissues. In particular, the aMTD-fused recombinant proteins according to one embodiment of the present invention may induce recombination of a target gene in the nucleus to influence greatly the investigation and identification of the function of the gene.

[0012] One aspect of the present invention relates to baseline platform that could be applied to unlimited number of designs, having cell-permeability applicable for biomedical sciences, preclinical and clinical studies that facilitate the traverse of biologically active macromolecules, including proteins, peptides, nucleic acids, chemicals and so on, across the plasma membrane in cells.

[0013] The present inventors analyzed, identified, and determined these critical factors that facilitate the cell permeable ability of aMTD sequences. These aMTD sequences are artificially assembled based on the critical factors (CFs) determined from in-depth analysis of previously published hydrophobic CPPs.

[0014] One aspect of the present invention relates to novel advanced macromolecule transduction domain (aMTD) sequences.

[0015] The aMTD sequences of one aspect of the present invention are the first artificially developed cell permeable polypeptides capable of mediating the transduction of biologically active macromolecules - including peptides, polypeptides, protein domains, or full-length proteins - through the plasma membrane of cells.

[0016] Another aspect of the present invention relates to the method of genetically engineering biologically active molecules having cell-permeability by fusing the aMTD sequences to the biologically active cargo molecules.

[0017] One aspect of the present invention also relates to its therapeutic application for the delivery of biologically active molecules to cells, involving cell-permeable recombinant proteins, where aMTDs are attached to the biologically active cargo molecules.

[0018] Another aspect of the present invention pertains to a method in which biologically active macromolecules are able to enter into live cells, as constructs of cell-permeable recombinant proteins comprised of aMTD sequences fused to biologically active macromolecules.

[0019] Other aspects of the present invention relate to an efficient use of aMTD sequences for molecule delivery, drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy, peptide therapy, gene delivery and so on.

[0020] Another aspect of the present invention relates to 240 new hydrophobic CPP sequences - aMTDs, determination of the aMTD-mediated intracellular delivery activity of the recombinant proteins, and comparison of the enhanced protein uptake by live cells at levels greater than or equal to the FGF4-derived MTS/MTM and HOURSS-derived MTD sequences. These strengths of newly invented aMTDs could address the setbacks on reference hydrophobic CPPs for clinical development and application. However, only those sequences identified in the claims are covered by the present invention.

[0021] One aspect of the present invention pertains to advanced macromolecule transduction domain (aMTD) sequences that transduce biologically active macromolecules into the plasma membrane.

[0022] Another aspect of the present invention directs to aMTD consisting of amino acid sequences having the following characteristics:

a.Amino acid length: 9 to 13

b.Bending potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end (12') of the sequence.

c.Rigidity/Flexibility: Instability Index (II): 40 to 60

d.Structural Feature: Aliphatic Index (AI): 180 to 220

e.Hydropathy: GRAVY: 2.1 to 2.6

f.Amino acid composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

[0023] According to one embodiment, the amino acid sequences have the general formula composed of 12 amino acid sequences as described below.

wherein (P) at the end of sequence (12') is proline, one of U sites is proline, X(s) and U(s) which is not proline are A, V, L and/or I.

[0024] According to one embodiment, the amino acid sequences having the general formula are selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO: 240, wherein only SEQ ID NO : 131 is claimed in combination with the cell-permeable Cre recombinant protein.

[0025] According to one embodiment, the secondary structure of the aMTD is α-Helix.

[0026] One aspect of the present invention further provides isolated polynucleotides that encode aMTD sequences described above.

[0027] According to one embodiment, the isolated polynucleotide is selected from the group consisting of SEQ ID NO : 241 to SEQ ID NO: 480, wherein only SEQ ID NO: 371 is claimed in combination with the cell-permeable Cre recombinant protein.

[0028] Described herein is also a method of identifying critical factors of aMTDs. The 6 methods comprise selecting superior hydrophobic CPPs from previously published reference hydrophobic CPPs; analyzing physiological and chemical characteristics of the selected hydrophobic CPPs; identifying features that are in association with cell-permeability out of these physiological and chemical characteristics; categorizing previously published reference hydrophobic CPPs into at least 2 groups and determining unique features by in-depth analysis of each groups of CPPs according to their cell-permeability and relative characteristics; configuring critical factors identified through analyzing the determined unique features; confirming the critical factors is valid through experimental studies; and determining critical factors that are based on the confirmed experimental studies.

[0029] The identified unique features are amino acid length, molecular weight, pI value, bending potential, rigidity, flexibility, structural feature, hydropathy, residue structure, amino acid composition and secondary structure.

[0030] The determined six critical factors consist of the following characteristics:

a.Amino Acid Length: 9 to 13

b.Bending Potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end of the sequence.

c.Rigidity/Flexibility: Instability Index (II): 40 to 60

d.Structural Feature: Aliphatic Index (AI): 180 to 220

e.Hydropathy: GRAVY: 2.1 to 2.6.

f.Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

g.Secondary structure: α-Helix

[0031] Further provided is a method of developing the aMTD sequences. The method comprises designing a platform of aMTDs having the below general formula described below;

wherein (P) at the end of sequence (12') is proline, one of U sites is proline, X(s) and U(s) which is not proline are A, V, L and/or I; and confirming whether a designed amino acid sequence satisfy six critical factors as follows:

a.Amino Acid Length: 9 to 13

b.Bending Potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end of the sequence.

c.Rigidity/Flexibility: Instability Index (II): 40 to 60

d.Structural Feature: Aliphatic Index (AI): 180 to 220

e.Hydropathy: GRAVY: 2.1 to 2.6.

f.Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

[0032] The six critical factors obtained the method of identifying unique features of aMTDs consist of the following factors:

a.Amino Acid Sequence: 12

b.Bending Potential: Proline (P) is positioned in the middle (5', 6', 7' or 8') and at the end (12') of the sequence.

c.Rigidity/Flexibility: Instability Index (II): 41.3 to 57.3

d.Structural Feature: Aliphatic Index (AI): 187.5 to 220

e.Hydropathy: GRAVY: 2.2 to 2.6.

f.Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

[0033] The secondary structure of the aMTD may be α-Helix.

[0034] According to one embodiment, the method further comprises developing the expression vectors of aMTD sequences fused to cargo proteins; selecting proper bacteria strain for inducible expression; purifying and preparing of aMTD-fused to cargo proteins in soluble form; and confirming their cell-permeability.

[0035] One aspect of present invention further provides isolated recombinant proteins with a cell-permeability. The isolated recombinant protein comprises an advanced macromolecule transduction domain (aMTD) sequences having amino acid sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO: 240, wherein only SEQ ID NO : 131 is claimed in combination with the cell-permeable Cre recombinant protein; and a biologically active molecule.

[0036] According to one embodiment, the biologically active molecules are any one selected from the group consisting of growth factors, enzymes, transcription factors, toxins, antigenic peptides, antibodies and antibody fragments.

[0037] According to one embodiment, the biologically active molecules are any one selected from the group consisting of enzymes, hormones, carriers, immunoglobulins, antibodies, structural proteins, motor functioning peptides, receptors, signaling peptides, storing peptides, membrane peptides, transmembrane peptides, internal peptides, external peptides, secreting peptides, virus peptides, native peptides, glycated proteins, fragmented proteins, disulfide bonded proteins, recombinant proteins, chemically modified proteins and prions.

[0038] According to one embodiment, the biologically active molecules are any one selected from the group consisting of nucleic acids, coding nucleic acid sequences, mRNAs, antisense RNA molecules, carbohydrates, lipids and glycolipids.

[0039] According to one embodiment, the biologically active molecules are at least one selected from the group consisting of biotherapeutic chemicals and toxic chemicals.

[0040] One aspect of the present invention further provides a method of genetically or epigenetically engineering and/or modifying biologically active molecules to have a cell-permeability. The method comprises fusing aMTDs to biologically active molecules under the optimized and effective conditions to generate biologically active molecules that can be cell-permeable, wherein the aMTD consists of any one of amino acid sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO: 240, wherein only SEQ ID NO : 131 is claimed.

[0041] One aspect of the present invention also pertains to cell-permeable recombinant protein for site-specific recombination based on advanced macromolecule transduction domain (aMTD) sequences capable of mediating the transduction of biologically active macromolecules into live cells.

[0042] Other aspect of the present invention relates to cell-/tissue-protein-based site-specific recombination based on an efficient use of aMTD sequences for protein delivery and recombinase delivery.

[0043] Described herein is an improved Cell-Permeable Cre (iCP-Cre) recombinant protein, which comprises a Cre protein and an advanced macromolecule transduction domain (aMTD) being composed of 9 to 13 amino acid sequences and having improved cell or tissue permeability,
wherein the aMTD is fused to one end or both ends of the Cre protein and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence; and

(c) having an instability index of 40 to 60; an aliphatic index of 180 to 220; and a grand average of hydropathy (GRAVY) of 2.1 to 2.6, as measured by Protparam.

[0044] According to one embodiment, one or more solubilization domain (SD)(s) are further fused to the end(s) of one or more of the Cre protein and the aMTD.

[0045] According to another embodiment, the aMTD may have α-Helix structure.

[0046] The aMTD may be composed of 12 amino acid sequences and represented by the following general formula:

wherein X(s) refers to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); one of U refers to proline and the other U(s) refer to A, V, L or I; and P refers to proline.

[0047] Another aspect of the present invention provides an iCP-Cre recombinant protein which is represented by any one of the following structural formula:

        A-B-C, A-C-B and A-C-B-C

wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell or tissue permeability, B is a Cre protein, and C is a solubilization domain (SD); and
the aMTD is composed of 9 to 13 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence;

(c) having an instability index of 40 to 60; an aliphatic index of 180 to 220; and a grand average of hydropathy (GRAVY) of 2.1 to 2.6, as measured by Protparam; and

(d) having α-Helix structure.

[0048] According to one embodiment of the present invention, the Cre protein may have an amino acid sequence of SEQ ID NO: 816.

[0049] According to another embodiment of the present invention, the Cre protein may be encoded by a polynucleotide sequence of SEQ ID NO: 817.

[0050] According to still another embodiment of the present invention, the Cre protein may further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

[0051] According to still another embodiment of the present invention, the aMTD may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240 wherein only SEQ ID NO: 131 is claimed.

[0052] According to still another embodiment of the present invention, the aMTD may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480 wherein only SEQ ID NO: 371 is claimed.

[0053] According to still another embodiment of the present invention, the SD(s), independently, may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 804.

[0054] According to still another embodiment of the present invention, the SD(s), independently, may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to 811.

[0055] According to still another embodiment of the present invention, the Cre recombinant protein may have one or more selected from a histidine-tag affinity domain and a clear localization sequence (NLS) additionally fused to one end thereof.

[0056] According to still another embodiment of the present invention, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812, and the NLS may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 834.

[0057] According to still another embodiment of the present invention, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813, and the NLS may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 815 and 835.

[0058] According to still another embodiment of the present invention, the fusion may be formed via a peptide bond or a chemical bond.

[0059] According to still another embodiment of the present invention, the iCP-Cre recombinant protein may be used for the production of a conditional knockout mouse.

[0060] Still another aspect of the present invention provides a polynucleotide sequence encoding the iCP-Cre recombinant protein.

[0061] According to one embodiment of the present invention, the polynucleotide sequence may be a polynucleotide sequence represented by SEQ ID NO: 819 or SEQ ID NO: 825.

[0062] According to another embodiment of the present invention, the polynucleotide sequence may be selected from the group consisting of SEQ ID NOs: 821, 827 and 831.

[0063] Still another aspect of the present invention provides a recombinant expression vector including the polynucleotide sequence.

[0064] Still another aspect of the present invention provides a transformant transformed with the recombinant expression vector.

[0065] Still another aspect of the present invention provides a preparing method of the iCP-Cre recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by the culturing.

[0066] Still another aspect of the present invention provides a composition including the iCP-Cre recombinant protein as an active ingredient.

[0067] According to one embodiment of the present invention, the composition may be used for the production of a conditional knockout mouse.

[0068] Still another aspect of the present invention provides use of the iCP-Cre recombinant protein for the production of a conditional knockout mouse.

[0069] Still another aspect of the present invention provides a method of producing a conditional knockout mouse, including preparing a mouse in which LoxP sites are located in both ends of a target gene; and administering to the mouse an effective amount of the iCP-Cre recombinant protein.

[0070] According to one embodiment of the present invention, the method is the administering is by portal vein or intrarenal injection.

[0071] Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Although a certain method and a material is described herein, it should not be construed as being limited thereto, any similar or equivalent method and material to those may also be used in the practice or testing of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0072] A "peptide" refers to a chain-type polymer formed by amino acid residues which are linked to each other via peptide bonds, and used interchangeably with "polypeptide." Further, a "polypeptide" includes a peptide and a protein.

[0073] Further, the term "peptide" includes amino acid sequences that are conservative variations of those peptides specifically exemplified herein. The term "conservative variation," as used herein, denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, nor leucine, or methionine for another, or substitution of one polar residue for another, for example, substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which may be substituted for one another include asparagine, glutamine, serine, and threonine.

[0074] The term "conservative variation" also includes use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides according to one embodiment of the present invention.

[0075] A person having ordinary skill in the art may make similar substitutions to obtain peptides having higher cell permeability and a broader host range. For example, one embodiment of the present invention provides peptides corresponding to amino acid sequence SEQ ID NO: 131 provided herein, as well as analogues, homologs, isomers, derivatives, amidated variations, and conservative variations thereof, as long as the cell permeability of the peptide remains.

[0076] Minor modifications to primary amino acid sequence of the peptides according to one embodiment of the present invention may result in peptides which have substantially equivalent or enhanced cell permeability, as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.

[0077] All peptides may be synthesized using L-amino acids, but D forms of all of the peptides may be synthetically produced. In addition, C-terminal derivatives, such as C-terminal methyl esters and C-terminal amidates, may be produced in order to increase the cell permeability of the peptide according to one embodiment of the present invention.

[0078] All of the peptides produced by these modifications are included herein, as long as in the case of amidated versions of the peptide, the cell permeability of the original peptide is altered or enhanced such that the amidated peptide is therapeutically useful. It is envisioned that such modifications are useful for altering or enhancing cell permeability of a particular peptide.

[0079] Furthermore, deletion of one or more amino acids may also result in a modification to the structure of the resultant molecule without any significant change in its cell permeability. This may lead to the development of a smaller active molecule which may also have utility. For example, amino- or carboxyl-terminal amino acids which may not be required for the cell permeability of a particular peptide may be removed.

[0080] The term "gene" refers to an arbitrary nucleic acid sequence or a part thereof having a functional role in protein coding or transcription, or regulation of other gene expression. The gene may be composed of all nucleic acids encoding a functional protein or a part of the nucleic acid encoding or expressing the protein. The nucleic acid sequence may include a gene mutation in exon, intron, initiation or termination region, promoter sequence, other regulatory sequence, or a unique sequence adjacent to the gene.

[0081] The term "primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA target polynucleotide and serves as the starting points for the stepwise synthesis of a polynucleotide from mononucleotides by the action of a nucleotidyltransferase as occurs, for example, in a polymerase chain reaction.

[0082] The term "coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a part thereof which encodes a particular gene product or a fragment thereof for which expression is desired, according to the normal base pairing and codon usage relationships. Coding sequences include exons in genomic DNA or immature primary RNA transcripts, which are joined together by the cellular biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of the nucleic acid, and the coding sequence may be deduced therefrom.

[0083] Described is an iCP-Cre recombinant protein, which comprises a Cre protein and an advanced macromolecule transduction domain (aMTD) being composed of 9 to 13 amino acid sequences, preferably 10 to 12 amino acid sequences, and having improved cell or tissue permeability,
wherein the aMTD is fused to one end or both ends of the Cre protein and has the following features of:

(a) being preferably composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acids, and preferably one or more of positions 5 to 8 and position 12 of its amino acid sequence; and

(c) having an instability index of preferably 40 to 60 and more preferably 41 to 58; an aliphatic index of preferably 180 to 220 and more preferably 185 to 225; and a grand average of hydropathy (GRAVY) of preferably 2.1 to 2.6 and more preferably 2.2 to 2.6 as measured by Protparam (see http://web.expasy.org/protparam/).

[0084] Further described is one or more solubilization domain (SD)(s) are further fused to one or more of the Cre protein and the aMTD, preferably one end or both ends of the Cre protein, and more preferably the C-terminus and the N- terminus of the Cre protein.

[0085] The aMTD may have α-Helix structure.

[0086] Further described is that the aMTD may be preferably composed of 12 amino acid sequences and represented by the following general formula:

wherein X(s) refers to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); one of U refers to proline and the other U(s) refer to A, V, L or I; and P refers to proline.

[0087] Described is also an iCP-Cre recombinant protein which is represented by any one of structural formula A-B-C, A-C-B and A-C-B-C, and preferably by A-B-C and A-C-B-C, and more preferably by A-C-B-C:

wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell or tissue permeability, B is a Cre protein, and C is a solubilization domain (SD); and

the aMTD is composed of 9 to 13, preferably 10 to 12 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence, and preferably, one or more of positions 5 to 8 and position 12 of its amino acid sequence;

(c) having an instability index of preferably 40 to 60 and more preferably 41 to 58; an aliphatic index of preferably 180 to 220 and more preferably 185 to 225; and a grand average of hydropathy (GRAVY) of preferably 2.1 to 2.6 and more preferably 2.2 to 2.6, as measured by Protparam (see http://web.expasy.org/protparam/); and

(d) preferably having α-Helix structure.

[0088] In one embodiment of the present invention, the Cre protein may have an amino acid sequence of SEQ ID NO: 816.

[0089] In another embodiment of the present invention, the Cre protein may be encoded by a polynucleotide sequence of SEQ ID NO: 817.

[0090] When the iCP-Cre recombinant protein is intended to be delivered to a particular cell, tissue, or organ, the Cre protein may form a fusion product, together with an extracellular domain of a ligand capable of selectively binding to a receptor which is specifically expressed on the particular cell, tissue, or organ, or monoclonal antibody (mAb) capable of specifically binding to the receptor or the ligand and a modified form thereof.

[0091] The binding of the peptide and a biologically active substance may be formed either by indirect linkage by a cloning technique using an expression vector at a nucleotide level or by direct linkage via chemical or physical covalent or non-covalent bond of the peptide and the biologically active substance.

[0092] In still another embodiment of the present invention, the Cre protein may preferably further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

[0093] The aMTD may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240. The aMTD may be preferably aMTD₂ of SEQ ID NO: 2, aMTD₆₁ of SEQ ID NO: 17, aMTD₁₆₅ of SEQ ID NO: 43, aMTD₂₆₄ of SEQ ID NO: 63, aMTD₅₆₃ of SEQ ID NO: 131, aMTD₅₈₂ of SEQ ID NO: 134, aMTD₅₈₅ of SEQ ID NO: 136, aMTD₆₂₃ of SEQ ID NO: 143, aMTD₆₆₁ of SEQ ID NO: 147, aMTD₈₄₇ of SEQ ID NO: 200, aMTD₈₈₈ of SEQ ID NO: 222 or aMTD₈₉₉ of SEQ ID NO: 229, wherein only aMTD₅₆₃ of SEQ ID NO: 131 is claimed herein.

[0094] The aMTD may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480. The aMTD may be preferably aMTD₂ encoded by a polynucleotide sequence of SEQ ID NO: 242, aMTD₆₁ encoded by a polynucleotide sequence of SEQ ID NO: 257, aMTD₁₆₅ encoded by a polynucleotide sequence of SEQ ID NO: 283, aMTD₂₆₄ encoded by a polynucleotide sequence of SEQ ID NO: 303, aMTD₅₆₃ encoded by a polynucleotide sequence of SEQ ID NO: 371, aMTD₅₈₂ encoded by a polynucleotide sequence of SEQ ID NO: 374, aMTD₅₈₅ encoded by a polynucleotide sequence of SEQ ID NO: 376, aMTD₆₂₃ encoded by a polynucleotide sequence of SEQ ID NO: 383, aMTD₆₆₁ encoded by a polynucleotide sequence of SEQ ID NO: 387, aMTD₈₄₇ encoded by a polynucleotide sequence of SEQ ID NO: 440, aMTD₈₈₈ encoded by a polynucleotide sequence of SEQ ID NO: 462 or aMTD₈₉₉ encoded by a polynucleotide sequence of SEQ ID NO: 469, wherein only aMTD₅₆₃ encoded by a polynucleotide sequence of SEQ ID NO: 371 is claimed herein.

[0095] In still another embodiment of the present invention, the SD(s) may have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798 to 804. The SD(s) may has one or more selected from the group consisting of SDA, SDB, SDB', SDC, SDD, SDE and SDF. The SD may be preferably SDA of SEQ ID NO: 798 and/or SDB of SEQ ID NO: 799, and more preferably SDA of SEQ ID NOs: 798 and SDB of SEQ ID NOs: 799 which has superior structural stability.

[0096] In still another embodiment of the present invention, the SDs may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805 to 811. The SD may be preferably SDA encoded by a polynucleotide sequence of SEQ ID NO: 805 and/or SDB encoded by a polynucleotide sequence of SEQ ID NO: 806, and more preferably, SDA and SDB having superior structural stability, which is encoded by a polynucleotide sequence of SEQ ID NOs: 805 and 806.

[0097] The iCP-Cre recombinant protein may be preferably selected from the group consisting of:

1) a recombinant protein, in which Cre protein having an amino acid sequence of SEQ ID NO: 816 is fused to the N-terminus or the C-terminus of aMTD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240, preferably SEQ ID NOs: 2, 17, 43, 63, 131, 134, 136, 143, 147, 200, 222 and 229, wherein only SEQ ID NO: 131 is claimed;

2) a recombinant protein, in which SD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 804, preferably SEQ ID NOs: 798, 799, 801, 802, 803, and 804, and more preferably SEQ ID NOs: 798 and 799, are further fused to the N-terminus or the C-terminus of the Cre protein in the recombinant protein of 1); and

3) described is also a recombinant protein, in which one or more of a histidine tag having an amino acid sequence of SEQ ID NO: 812 and a NLS having an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 834 are further fused to the N-terminus or the C-terminus of the aMTD in the recombinant protein of 1) or 2).

[0098] The Cre protein is delivered into the cells or nucleus, and the Cre protein recognizes LoxP sites of DNA to remove a target gene that exist between two LoxP sites, resulting in inactivation of the gene (Cre/LoxP system).

[0099] The recombinant expression vector may include a tag sequence which makes it easy to purify the recombinant protein, for example, consecutive histidine codon, maltose binding protein codon, Myc codon, etc., and further include a fusion partner to enhance solubility of the recombinant protein, etc. Further, for the overall structural and functional stability of the recombinant protein or flexibility of the proteins encoded by respective genes, the recombinant expression vector may further include one or more glycine, proline, and spacer amino acid or polynucleotide sequences including AAY amino acids. Furthermore, the recombinant expression vector may include a sequence specifically digested by an enzyme in order to remove an unnecessary region of the recombinant protein, an expression regulatory sequence, and a marker or reporter gene sequence to verify intracellular delivery, but is not limited thereto.

[0100] In still another embodiment of the present invention, the iCP-Cre recombinant protein may preferably have a one or more of a histidine-tag affinity domain and a nuclear localization sequence (NLS) additionally fused to one end thereof. Preferably, the histidine-tag or the NLS may be fused to the N-terminus of the Cre protein, and more preferably, both of the histidine-tag and the nuclear localization sequence may be fused to the N-terminus of the Cre protein.

[0101] In still another embodiment of the present invention, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812, and the NLS may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 834. The NLS may has one selected from the group consisting of NLS-1 and NLS-2.

[0102] In still another embodiment of the present invention, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813, and the NLS may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 815 and 835.

[0103] In still another embodiment of the present invention, the fusion may be formed via a peptide bond or a chemical bond.

[0104] The chemical bond may be preferably selected from the group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds, and covalent bonds.

[0105] According to still another embodiment of the present invention, the iCP-Cre recombinant protein may be used for the production of a conditional knockout mouse.

[0106] The term "conditional knockout" (or "conditional gene knockout") refers to eliminate a specific gene in a certain cell/tissue, and expression or the gene is suppressed. The term "conditional knockout mouse" refers to mouse which carries one or more genetic manipulations leading to deactivation of a target gene in a tissue and optionally time specific manner.

[0107] The conditional gene knockout which the gene expression limited at specific times differs from traditional gene knockout which the gene was deleted from beginning of life. The most commonly used technique is the Cre/LoxP recombination system for conditional knockout mouse. The Cre/LoxP recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific gene in the DNA of cells. The system consists of a single enzyme, Cre (cyclization recombinase), that recombines a pair of short target sequences called the LoxP sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre and the original Lox (loci of recombination) site called the LoxP sequence are derived from bacteriophage P1. The Cre protein specifically recognizes two LoxP sites within DNA and causes recombination between them. During recombination two strands of DNA exchange information. This recombination will cause a deletion of the genes between the two LoxP sites, depending on their orientation. An entire gene can be removed to inactivate it. Only a few cell types express Cre protein and no mammalian cells express it so there is no risk of accidental activation of LoxP sites when using conditional gene knockout in mammals.

[0108] Still another aspect of the present invention provides a polynucleotide sequence encoding the iCP-Cre recombinant protein.

[0109] The polynucleotide sequence according to one embodiment of the present invention may be present in a vector in which the polynucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the polynucleotide sequence by a suitable host cell.

[0110] According to one embodiment of the present invention, the polynucleotide sequence may be selected from the following groups:

1) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480, preferably SEQ ID NOs: 242, 257, 283, 303, 371, 374, 376, 383, 387, 440, 462 and 469, and wherein SEQ ID NO: 371 is claimed, is operably linked with a polynucleotide sequence of SEQ ID NO: 817; and

2) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to 811, preferably SEQ ID NOs: 805, 806, 808, 809, 810, and 811, and more preferably SEQ ID NOs: 805 and/or 806 is further operably linked to the polynucleotide sequence of 1).

[0111] Within the expression vector, the term "operably linked" is intended to mean that the polynucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the polynucleotide sequence. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements. Such operable linkage with the expression vector can be achieved by conventional gene recombination techniques known in the art, while site-directed DNA cleavage and linkage are carried out by using conventional enzymes known in the art.

[0112] The expression vectors may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector, and may further include a polynucleotide sequence that enables the vector to replicate in the host cell in question.

[0113] The expression vector constructed according to the present invention may be the vector where the polynucleotide encoding the iCP-Cre recombinant protein (where an aMTD is fused to the N-terminus or C-terminus of a Cre protein) is inserted within the multiple cloning sites (MCS), preferably Ndel/EcoRI or SalI/XhoI site of a pET-28a(+) vector (Novagen, USA).

[0114] In still another embodiment of the present invention, the polynucleotide encoding the SD being additionally fused to the N-terminus or C-terminus of a Cre protein may be inserted into a cleavage site of restriction enzyme (Ndel, EcoRI, SalI, Xhol, etc.) within the multiple cloning sites (MCS) of a pET-28a(+) vector (Novagen, USA).

[0115] In still another embodiment of the present invention, the polynucleotide is cloned into a pET-28a(+) vector bearing a NLS residues to the N-terminus of the iCP-Cre recombinant protein to allow efficient nuclear transport.

[0116] In still another embodiment of the present invention, the polynucleotide is cloned into a pET-28a(+) vector bearing a His-tag sequence so as to fuse six histidine residues to the N-terminus of the iCP-Cre recombinant protein to allow easy purification.

[0117] According to one embodiment of the present invention, the polynucleotide sequence may be a polynucleotide sequence represented by SEQ ID NO: 819 or SEQ ID NO: 825.

[0118] According to another embodiment of the present invention, the polynucleotide sequence may be further fused with SD, and may be represented by a polynucleotide sequence represented by SEQ ID NOs: 821, 827 and 831.

[0119] The polynucleotide sequence may be fused with a histidine-tag affinity domain and NLS, and may be a polynucleotide sequence of SEQ ID NOs: 823, 829 and 833.

[0120] Preferably, the iCP-Cre recombinant protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 820, 826 and 830.

[0121] Still another aspect of the present invention provides a recombinant expression vector including the polynucleotide sequence.

[0122] Preferably, the vector may be inserted in a host cell and recombined with the host cell genome, or refers to any nucleic acid including a nucleotide sequence competent to replicate spontaneously as an episome. Such a vector may include a linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc.

[0123] Preferably, the vector may be genetically engineered to incorporate the nucleic acid sequence encoding the recombinant protein in an orientation either N-terminal and/or C-terminal to a nucleic acid sequence encoding a peptide, a polypeptide, a protein domain, or a full-length protein of interest, and in the correct reading frame so that the recombinant protein consisting of aMTD, Cre protein, and preferably SD may be expressed. Expression vectors may be selected from those readily available for use in prokaryotic or eukaryotic expression systems.

[0124] Standard recombinant nucleic acid methods may be used to express a genetically engineered recombinant protein. The nucleic acid sequence encoding the recombinant protein according to one embodiment of the present invention may be cloned into a nucleic acid expression vector, e.g., with appropriate signal and processing sequences and regulatory sequences for transcription and translation, and the protein may be synthesized using automated organic synthetic methods. Synthetic methods of producing proteins are described in, for example, the literature [Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis by Gregg B. Fields (Editor), Sidney P. Colowick, Melvin I. Simon (Editor), Academic Press (1997)].

[0125] In order to obtain high level expression of a cloned gene or nucleic acid, for example, a cDNA encoding the recombinant protein according to one embodiment of the present invention, the recombinant protein sequence may be typically subcloned into an expression vector that includes a strong promoter for directing transcription, a transcription/translation terminator, and in the case of a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and are described, e.g., in the literature [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N.Y. (2001); and Ausube, et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N. Y. (1989)]. Bacterial expression systems for expression of the recombinant protein according to one embodiment of the are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector may be preferably an adenoviral vector, an adeno-associated vector, or a retroviral vector.

[0126] Generally, the expression vector for expressing the cell permeable recombinant protein according to one embodiment of the present invention in which the cargo protein, i.e. Cre protein, is attached to the N-terminus, C-terminus, or both termini of aMTD may include regulatory sequences including, for example, a promoter, operably attached to a sequence encoding the advanced macromolecule transduction domain. Non-limiting examples of inducible promoters that may be used include steroid-hormone responsive promoters (e.g., ecdysone-responsive, estrogen-responsive, and glutacorticoid-responsive promoters), tetracycline "Tet-On" and "Tet-Off" systems, and metal-responsive promoters.

[0127] The recombinant protein may be introduced into an appropriate host cell, e.g., a bacterial cell, a yeast cell, an insect cell, or a tissue culture cell. The recombinant protein may also be introduced into embryonic stem cells in order to generate a transgenic organism. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available for generating the recombinant protein.

[0128] Known methods may be used to construct vectors including the polynucleotide sequence according to one embodiment of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. For example, these techniques are described in the literature [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N. Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience, N.Y. (1989)].

[0129] Still another aspect of the present invention provides a transformant transformed with the recombinant expression vector.

[0130] The transformation includes transfection, and refers to a process whereby a foreign (extracellular) DNA, with or without an accompanying material, enters into a host cell. The "transfected cell" refers to a cell into which the foreign DNA is introduced into the cell, and thus the cell harbors the foreign DNA. The DNA may be introduced into the cell so that a nucleic acid thereof may be integrated into the chromosome or replicable as an extrachromosomal element. The cell introduced with the foreign DNA, etc. is called a transformant.

[0131] As used herein, 'introducing' of a protein, a peptide, an organic compound into a cell may be used interchangeably with the expression of 'carrying,' 'penetrating,' 'transporting,' 'delivering,' 'permeating' or 'passing.'

[0132] It is understood that the host cell refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced, and refers not only to the particular subject cell but also to the progeny or potential progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0133] The host cells may be preferably bacterial cells, and as the bacterial cells, there are, in principle, no limitations. They may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest, preferably for site-specific integration, and they may be cultured on a manufacturing scale. Preferably, the host cells may have the property to allow cultivation to high cell densities.

[0134] Examples of bacterial host cells that may be used in the preparation of the recombinant protein are E. coli (Lee, 1996; Hannig and Makrides, 1998), Bacillus subtilis, Pseudomonas fluorescens (Squires et al., 2004; Retallack et al., 2006) as well as various Corynebacterium (US 2006/0003404 A1) and Lactococcus lactis (Mierau et al., 2005) strains. Preferably, the host cells are Escherichia coli cells.

[0135] More preferably, the host cell may include an RNA polymerase capable of binding to a promoter regulating the gene of interest. The RNA polymerase may be endogenous or exogenous to the host cell.

[0136] Preferably, host cells with a foreign strong RNA polymerase may be used. For example, Escherichia coli strains engineered to carry a foreign RNA polymerase (e.g. like in the case of using a T7 promoter a T7-like RNA polymerase in the so-called "T7 strains") integrated in their genome may be used. Examples of T7 strains, e.g. BL21(DE3), HMS174(DE3), and their derivatives or relatives (see Novagen, pET System manual, 11th edition), may be widely used and commercially available. Preferably, BL21-CodonPlus (DE3)-RIL or BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) may be used. These strains are DE3 lysogens containing the T7 RNA polymerase gene under control of the lacUV5 promoter. Induction with IPTG allows production of T7 RNA polymerase which then directs the expression of the gene of interest under the control of the T7 promoter.

[0137] The host cell strains, E. coli BL21(DE3) or HMS174(DE3), which have received their genome-based T7 RNA polymerase via the phage DE3, are lysogenic. It is preferred that the T7 RNA polymerase contained in the host cell has been integrated by a method which avoids, or preferably excludes, the insertion of residual phage sequences in the host cell genome since lysogenic strains have the disadvantage to potentially exhibit lytic properties, leading to undesirable phage release and cell lysis.

[0138] Still another aspect of the present invention provides a preparing method of the iCP-Cre recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by culturing.

[0139] Culturing may be preferably in a mode that employs the addition of a feed medium, this mode being selected from the fed-batch mode, semi-continuous mode, or continuous mode. The bacterial expression host cells may include a DNA construct which is integrated in their genome and carrying the DNA sequence encoding the protein of interest under the control of a promoter that enables expression of said protein.

[0140] There are no limitations in the type of the culture medium. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids), or it may be chemically defined, without any complex compounds. Preferably, a defined medium may be used. The defined media (also called minimal or synthetic media) are exclusively composed of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins, and, in view of a possible strain auxotrophy, specific amino acids or other substances such as thiamine. Most preferably, glucose may be used as a carbon source. Usually, the carbon source of the feed medium serves as the growth-limiting component which controls the specific growth rate.

[0141] Host cells may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. The literature [Scopes, Protein Purification: Principles and Practice, New York: Springer-Verlag (1994)] describes a number of general methods for purifying recombinant (and non-recombinant) proteins. The methods may include, e.g., ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, selective precipitation, dialysis, and hydrophobic interaction chromatography. These methods may be adapted to devise a purification strategy for the cell permeable recombinant protein. If the cell permeable recombinant protein includes a purification handle, such as an epitope tag or a metal chelating sequence, affinity chromatography may be used to easily purify the protein.

[0142] The amount of the protein produced may be evaluated by detecting the advanced macromolecule transduction domain directly (e.g., using Western analysis) or indirectly (e.g., by assaying materials derived from the cells for specific DNA binding activity, such as by electrophoretic mobility shift assay). Proteins may be detected prior to purification, during any stage of purification, or after purification. In some implementations, purification or complete purification may not be necessary.

[0143] The genetically engineered recombinant protein prepared by the method according to one embodiment of the present invention may be a cell/tissue-permeable protein. In particular, it may be removing part or all of a target gene in the nucleus to inactivate the gene.

[0144] The cell permeable protein prepared by the method according to one embodiment of the present invention may be used for the production of a conditional knockout mouse in which activity of a target gene is inhibited.

[0145] The cell permeable recombinant proteins according to one embodiment of the present invention may be used in vitro to investigate protein function or may be used to maintain cells in a desired state.

[0146] Still another aspect of the present invention provides a composition including the iCP-Cre recombinant protein as an active ingredient.

[0147] The composition may be administered to a mouse to produce a conditional knockout mouse in which a target gene is inactivated. The composition may preferably comprise the active ingredient in an amount of 0.1 to 99.9% by weight, based on the total weight of the composition. In addition to the above active ingredient, the composition may comprise a buffer, an ajuvant, etc. which is physiologically acceptable while stabilizing the recombinant protein.

[0148] Still another aspect of the present invention provides use of the iCP-Cre recombinant protein for the production of a conditional knockout mouse.

[0149] Still another aspect of the present invention provides a method of producing a conditional knockout mouse, including preparing a mouse in which LoxP sites are located in both ends of a target gene; and administering to mouse an effective amount of the iCP-Cre recombinant protein.

[0150] The mouse is a transgenic mouse, in which two LoxP sites exist at both ends or in the exon region of the target gene. In the absence of Cre protein, the target gene is expressed. However, in the presence of Cre protein, it recognizes the LoxP sites to remove the target gene, thereby suppressing expression of the gene. Therefore, when an effective amount of the Cre recombinant protein is administered to the mouse, conditional knockout of the target gene occurs. The target gene expression may be examined at an mRNA level or at a protein level.

[0151] In the preparation method of the conditional knockout mouse, the composition including the iCP-Cre recombinant protein as an active ingredient may be administered to the mouse in a common mode of administration via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, percutaneous, topical, intraocular, or intradermal route, and preferably, via intraperitoneal or intravenous route.

[0152] In the method, the administering is by portal vein injection or intrarenal injection.

[Advantageous Effects]

[0153] One aspect of the present invention provides artificially constructed aMTD sequences based on the critical factors (CFs) that overcome the limitations of prior arts (MTM/MTS/MTD), such as limited diversity and unpredictable cell-permeability. Based on the CFs that assure the cell-permeability, the aMTD displays these sequences shows up to 109.9 relative fold enhanced ability compared to prior arts thereof to deliver biologically active macromolecules into live cells. Therefore, according to one aspect of the present invention, the aMTD/SD are fused to the Cre protein to provide an iCP-Cre recombinant protein showing improved cell/tissue-permeability and intranuclear delivery, and enhanced protein solubility and yield.

[0154] This iCP-Cre recombinant protein with improved cell/tissue-permeability may mediate conditional knockout of a target gene in the nucleus at a particular period by the Cre/LoxP system in vivo and in vitro. By applying the iCP-Cre recombinant protein to a mouse, a conditional knockout mouse in which the target gene is inactivated may be produced. Thus, the iCP-Cre recombinant protein according to one embodiment of the present invention may be utilized to study of the function and action of the gene.

[Description of Drawings]

[0155] 

FIG. 1 shows Structure of aMTD- or rPeptide-Fused Recombinant Proteins. A schematic diagram of the His-tagged CRA recombinant proteins is illustrated and constructed according to the present invention. The his-tag for affinity purification (white), aMTD or rPeptide (gray) and cargo A (CRA, black) are shown.

FIGs. 2a to 2c show Construction of Expression Vectors for aMTDs- or rPeptide-Fused Recombinant Proteins. These FIGs. show the agarose gel electrophoresis analysis showing plasmid DNA fragments at 645bp insert encoding aMTDs or rPeptide-fused CRA cloned into the pET28a(+) vector according to the present invention.

FIGs. 3a to 3d show Inducible Expression of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant aMTD- or random peptide-fused CRA recombinant proteins were transformed in E. coli BL21 (DE3) strain. Expression of recombinant proteins in E.coli before (-) and after (+) induction with IPTG was monitored by SDS-PAGE, and stained with Coomassie blue.

FIGs. 4a and 4b show Purification of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant proteins were purified by Ni² + affinity chromatography under the natural condition. Purification of recombinant proteins displayed through SDS-PAGE analysis.

FIGs. 5a to 5u show Determination of aMTD-Mediated Cell-Permeability. Cell-permeability of a negative control (A: rP38) and reference hydrophobic CPPs (MTM12 and MTD85) are shown. The cell-permeability of each aMTD and/or rPeptide is visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins (HMCA) fused to negative control (rP38), reference CPP (MTM12 or MTD85) or new hydrophobic CPP (aMTD) are shown with light thick line and indicated by arrows.

FIGs. 6a to 6c show Determination of rPeptide-Mediated Cell-Permeability. The cell-permeability of each aMTD and/or rPeptide was visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins fused to rPeptides are shown with light thick line and indicated by arrows.

FIGs. 7a to 7k shows Visualized Cell-Permeability of aMTD-Fused Recombinant Proteins. NIH3T3 cells were treated with FITC-labeled protein (10 uM) fused to aMTD for 1 hour at 37°C. Cell-permeability of the proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIGs. 8a to 8b show Visualized Cell-Permeability of rPeptide-Fused Recombinant Proteins. Cell-permeability of rPeptide-fused recombinant proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIGs. 9a to 9c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Negative Control (rP38). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a negative control (A: rP38).

FIGs. 10a to 10c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTM12). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTM12).

FIGs. 11a to 11c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTD85). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTD85).

FIGs. 12 shows Relative Cell-Permeability of rPeptide-Mediated Recombinant Proteins Compared to Average that of aMTDs. The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to rPeptides and that (average value: aMTD AVE) of aMTDs.

FIGs. 13a and 13b show Association of Cell-Permeability with Amino Acid Composition in aMTD Sequences. These graphs display delivery potential (Geometric Mean) of aMTDs influenced with amino acid composition (A, I, V and L).

FIGs. 14a and 14b show Association of Cell-Permeability with Critical Factors in aMTDs. These graphs show the association of cell-permeability with critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIGs. 15a and 15b show Relative Relevance of aMTD-Mediated Cell-Permeability with Critical Factors. Cell-permeability of 10 high and 10 low ranked aMTDs in their delivery potential were examined for their association with the critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIG. 16 shows Relative Relevance of rPeptide-Mediated Cell-Permeability with Hydropathy Range (GRAVY). This graph and a chart illustrate relative relevance of rPeptide-mediated cell-permeability with its hydropathy range (GRAVY).

FIG. 17 shows Structure of Cre Recombinant Proteins. A schematic diagram of the aMTD/SD-fused Cre recombinant proteins having cell-permeability is illustrated and constructed according to the present invention.

FIG. 18 shows agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding aMTD/SD-fused Cre cloned into the pET28a (+) vector according to example <6-1> .

FIG. 19 shows Inducible Expressions and Purifications of Cre Recombinant Proteins and Solubility/Yield of Purified Cre Recombinant Proteins according to Example <6-3>. Recombinant proteins were transformed in E. coli BL21 (DE3) CodonPlus-RIL strain. The cloned recombinant proteins have confirmed the inducible expression through SDS-PAGE Analysis. The confirmed proteins purified by affinity chromatography with Nickel Resin or Cobalt (II) Resin.

FIG. 20 shows Structure of Cre Recombinant Proteins. A schematic diagram of the aMTD/SD-fused Cre recombinant proteins having cell-permeability and control protein without aMTD is illustrated and constructed according to the present invention.

FIG. 21 shows agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding Cre lacking aMTD and/or SD cloned into the pET28a (+) vector according to example <6-1>.

FIG. 22 shows Inducible Expressions and Purifications of Cre Recombinant Proteins according to example <6-3>. Recombinant proteins were transformed in E. coli BL21 (DE3) CoconPlus-RIL strain. The cloned recombinant proteins and control proteins have confirmed the inducible expression through SDS-PAGE Analysis. The confirmed proteins purified by affinity chromatography with Cobalt (II) Resin.

FIG. 23 shows Biological Activity of Cre Recombinant protein with Linearized Substrate Containing LoxP Sites according to example <7-1>. Functional activity of iCP-Cre was determined by a substrate (NEB) that contains LoxP-floxed ampicillin resistance gene. The iCP-Cre (0.1 ug) or NEB Cre (0.2 ug) were incubated with the substrate (150 ng), and then, the number of colonies were analyzed after the transformation.

FIG. 24 shows Biological Activity of Cre Recombinant protein with Circular Substrate Containing LoxP Sites according to example <7-2>. Functional activity of iCP-Cre was determined by the constructed substrate that contains ampicillin resistance gene and stop sequence floxed by LoxP. The iCP-Cre (0.1 ug) or NEB Cre (0.2 ug) was incubated with the substrate (150 ng), and the number of colonies were analyzed after the transformation.

FIG. 25 shows Structure of Cre Recombinant Proteins fused various aMTDs. A schematic diagram of the improved cell-permeable Cre (iCP-Cre) recombinant proteins fused various aMTDs having cell-permeability are illustrated and constructed according to the present invention.

FIG. 26 shows the agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding various aMTDs-used Cre cloned into the pET28a (+) vector according to example <8-1>.

FIG. 27 shows Solubility/Yield of Purified Cre Recombinant Proteins fused various aMTDs according to example <8-1>.

FIG. 28a shows aMTD-Mediated Cell-Permeability of Cre Recombinant Proteins fused various aMTDs according to example <8-2>. RAW 264.7 cells were exposed to FITC-labeled Cre recombinant proteins (10 uM) for 1 hour, treated with proteinase K to remove cell-associated but non-internalized proteins and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.

FIG. 28b shows aMTD-Mediated Cell-Permeability of Cre Recombinant Proteins fused various aMTDs according to example <8-2>. Gray shaded area represents untreated RAW 264.7 cells (vehicle); each of the lines represents FITC-fused cells (FITC only); His-tagged recombinant proteins lacking aMTD and/or SDs (HNC and HNACB); and His-tagged recombinant proteins fused various aMTDs (HNM_#ACB) from the left.

FIG. 29 shows shows Biological Activity of aMTD/SD-fused Cre Recombinant Proteins fused various aMTDs according to example <8-3>.

FIG. 30 shows aMTD-Mediated Cell-Permeability of aMTD/SD-fused Cre Recombinant Proteins according to example <9-1>. RAW 264.7 cells were exposed to FITC-labeled Cre recombinant proteins (10 M) for 1 hour, treated with proteinase K to remove cell-associated but non-internalized proteins and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.

FIG. 31 shows aMTD-Mediated Intracellular Localization and Intranuclear Localization of aMTD/SD-Fused Cre Recombinant Proteins according to example <9-2>.

FIG. 32 shows Tissue Distribution of aMTD/SD-Fused Cre Recombinant Proteins in vivo according to example 10.

FIG. 33 shows aMTD-Mediated Cell-To-Cell Delivery according to example 11. RAW 264.7 cells exposed to 10 uM FITC-HNACB or FITC-HNM₅₆₃ACB for 2 hours, were mixed with non-treated RAW 264.7 cells pre-stained with Cy5.5 labeled anti-CD14 antibody, and analyzed by flow cytometry (left, top). The top (right) panel shows a mixture of double negative cells (cells exposed to FITC- HNACB (Non-CP-Cre) that did not incorporate the protein) and single positive Cy5.5 labeled cells; whereas, second panel from the left contains FITC-Cy5.5 double-positive cells generated by the transfer of FITC-HNM₅₆₃ACB (iCP-Cre) to Cy5.5 labeled cells and the remaining FITC and Cy5.5 single-positive cells. The bottom panels show FITC fluorescence profiles of cell populations before mixing (coded as before) and 1 hour after the same cells were mixed with Cy5.5-labeled cells.

FIG. 34 shows Biological Activity of iCP-Cre Recombinant Proteins for Dose Dependency with Circular Substrate Containing LoxP Sites according to example 12. The iCP-Cre (1, 10, 100, 200, 500, 1000 ng) were incubated with the substrate (150 ng), and the number of colonies were analyzed after the transformation.

FIG. 35 shows Biological Activity of iCP-Cre Recombinant Proteins with Color-Switch Reporter Cell Line Containing LoxP Sites according to example 13. Tex.loxp.EG is a T-lymphocyte line in which Cre-mediated recombination activates the expression of a green fluorescent protein (GFP) reporter gene. Tex.loxP.EG cells exposed to 10 uM iCP-Cre for 2 hours with serum-free RPMI, and after 24 hours, GFP expression levels were analyzed by flow cytometry. Untreated cells (gray) were served as control.

FIG. 36 shows Systemic Recombination Activity of iCP-Cre with ROSA26-LSL-LacZ Mice according to example <14-1>. ROSA26-LSL-LacZ reporter mice are in a transgenic line that Cre-mediated recombination activates the expression of β-galactosidase, and blue color being displayed when X-gal staining. ROSA26-LSL-LacZ mice were injected with 12 mg/kg/dayiCP-Cre or with a buffer control intravenously injection for five consecutive days and sacrificed 2 days later. The indicated organs were removed, stained with X-Gal, and sectioned at 20 um. Tissues from ROSA26-LSL-LacZ mice, which constitutively express lacZ, were analyzed.

FIG. 37 shows Systemic Recombination Activity of iCP-Cre with ROSA26-LSL-EYFP Mice according to example <14-2>. ROSA26-LSL-EYFP reporter mice are in a transgenic line that Cre-mediated recombination activates the expression of enhanced yellow fluorescence protein (eYFP). ROSA26-LSL-EYFP mice were injected with 12 mg/kg/day iCP-Cre or with a buffer control intravenously injection for five consecutive days and sacrificed 2 days later. The indicated organs were removed and sectioned at 20 uM. Tissues from ROSA26-LSL-EYFP mice, which constitutively express eYFP, were detected by fluorescent microscope.

FIG. 38 shows Systemic Recombination Activity of iCP-Cre with SOCS3^f/f Conditional Knockout Mice according to example <14-3>. SOCS3^f/fmice were injected with 12 mg/kg/day iCP-Cre or with a buffer control intravenously injction for five consecutive days and sacrificed 2 days later. The indicated organs were removed, and mRNA was isolated. RT-PCR was carried out to analyze a reduction in the SOCS3 mRNA expression.

FIG. 39 shows in vivo Systemic Recombination Activity of iCP-Cre recombinant proteins with SOCS3^f/f Conditional Knockout Mice according to example <14-3>. SOCS3^f/f mice were injected with 12 mg/kg/day iCP-Cre or with a buffer control intravenously injection for five consecutive days and sacrificed 2 days later. The indicated organs were removed, proteins were isolate. Western blot analysis was carried out using the tissues to analyze a reduction in the SOCS3 protein expression.

FIG. 40 shows in vivo Systemic Recombination Activity of iCP-Cre Recombinant Proteins for Dose Dependency with SOCS3^f/f Conditional Knockout Mice according to example <14-3>. SOCS3^f/f mice were injected with 1, 2, 4, 6, 10 mg/kg/day iCP-Cre recombinant proteins or with a buffer control intravenously injection for five consecutive days and sacrificed 2 days later. The indicated organs were removed, and mRNA was isolated. RT-PCR was carried out to analyze a reduction in the SOCS3 mRNA expression.

FIG. 41 shows Systemic Recombination Activity of iCP-Cre Recombinant Proteins for Dose Dependency with SOCS3^f/f Conditional Knockout Mice according to example <14-3>. SOCS3^f/f mice were injected with 1, 2, 4, 6, 10 mg/kg/day iCP-Cre recombinant proteins or with a buffer control intravenously for five consecutive days and sacrificed 2 days later. The indicated organs were removed, proteins were isolate. Western blot analysis was carried out using the tissues to analyze a reduction in the SOCS3 protein expression.

FIG. 42 shows Organ-Specific Recombination Activity of iCP-Cre Recombinant Proteins with SOCS3^f/f Conditional Knockout Mice Treated by Local Administration according to example <14-3>. SOCS3^f/f mice were injected with 4 mg/kg/day iCP-Cre recombinant proteins or with a buffer control by portal vein injection or intrarenal injection. The indicated organs were removed, and mRNA was isolated. RT-PCR was carried out to analyze a reduction in the SOCS3 mRNA expression.

FIG. 43 shows Systemic Recombination Activity of iCP-Cre Recombinant Proteins with ROSA^nT-nG Mice according to example <14-4>. ROSA^nT-nG reporter mice are transgenic mice that contain a transgene encoding an enhanced tandem dimer tomato red fluorescent protein (tdTomato Red) in the ROSA26 locus with a lox-transcriptional stop-lox cassette (LSL) inserted proximal to the transcriptional start site, and Cre-mediated recombination activates the expression of enhanced green fluorescence protein (eGFP). The mice were injected with 12 mg/kg/day iCP-Cre recombinant proteins or with a buffer control intravenously for five consecutive days and sacrificed 2 days later. The indicated organs were removed, and mRNA was isolated. RT-PCR was carried out to analyze a reduction in the SOCS3 mRNA expression.

[Mode for Invention]

1. Analysis of Reference Hydrophobic CPPs to Identify 'Critical Factors' for Development of Advanced MTDs

[0156] Previously reported MTDs were selected from a screen of more than 1,500 signal peptide sequences. Although the MTDs that have been developed did not have a common sequence or sequence motif, they were all derived from the hydrophobic (H) regions of signal sequences (HOURSS) that also lack common sequences or motifs except their hydrophobicity and the tendency to adopt alpha-helical conformations. The wide variation in H-region sequences may reflect prior evolution for proteins with membrane translocating activity and subsequent adaptation to the SRP/Sec61 machinery, which utilizes a methionine-rich signal peptide binding pocket in SRP to accommodate a wide-variety of signal peptide sequences.

[0157] Previously described hydrophobic CPPs (e.g. MTS/MTM and MTD) were derived from the hydrophobic regions present in the signal peptides of secreted and cell surface proteins. The prior art consists first, of ad hoc use of H-region sequences (MTS/MTM), and second, of H-region sequences (with and without modification) with highest CPP activity selected from a screen of 1,500 signal sequences (MTM). Second prior art, the modified H-region derived hydrophobic CPP sequences had advanced in diversity with multiple number of available sequences apart from MTS/MTM derived from fibroblast growth factor (FGF) 4. However, the number of MTDs that could be modified from naturally occurring secreted proteins are somewhat limited. Because there is no set of rules in determining their cell-permeability, no prediction for the cell-permeability of modified MTD sequences can be made before testing them.

[0158] The hydrophobic CPPs, like the signal peptides from which they originated, did not conform to a consensus sequence, and they had adverse effects on protein solubility when incorporated into protein cargo. We therefore set out to identify optimal sequence and structural determinants, namely critical factors (CFs), to design new hydrophobic CPPs with enhanced ability to deliver macromolecule cargoes including proteins into the cells and tissues while maintaining protein solubility. These newly developed CPPs, advanced macromolecule transduction domains (aMTDs) allowed almost infinite number of possible designs that could be designed and developed based on the critical factors. Also, their cell-permeability could be predicted by their character analysis before conducting any in vitro and/or in vivo experiments. These critical factors below have been developed by analyzing all published reference hydrophobic CPPs.

1-1. Analysis of Hydrophobic CPPs

[0159] Seventeen different hydrophobic CPPs (Table 1) published from 1995 to 2014 (Table 2) were selected. After physiological and chemical properties of selected hydrophobic CPPs were analyzed, 11 different characteristics that may be associated with cell-permeability have been chosen for further analysis. These 11 characteristics are as follows: sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition and secondary structure of the sequences (Table 3).

[0160] Table 1 shows the Summary of Published Hydrophobic Cell-Penetrating Peptides which were Chosen.

[Table 1]

#	Pepides	Origin	Protein	Ref.
1	MTM	Homo sapiens	NP_001998 Kaposi fibroblast growth factor (K-FGF)	1
2	MTS	Homo sapiens	NP_001998 Kaposi fibroblast growth factor (K-FGF)	2
3	MTD10	Streptomyces coelicolor	NP_625021 Glycosyl hydrolase	8
4	MTD13	Streptomyces coelicolor	NP_639877 Putative secreted protein	3
5	MTD47	Streptomyces coelicolor	NP_627512 Secreted protein	4
6	MTD56	Homo sapiens	P23274 Peptidyl-prolyl cis-trans isomerase B precursor	5
7	MTD73	Drosophila melanogaster	AAA17887 Spatzle (spz) protein	5
8	MTD77	Homo sapiens	NP_003231 Kaposi fibroblast growth factor (K-FGF)	6
9	MTD84	Phytophthora cactorum	AAK63068 Phytotoxic protein PcF precusor	4
10	MTD85	Streptomyces coelicolor	NP_629842 Peptide transport system peptide binding protein	7
11	MTD86	Streptomyces coelicolor	NP_629842 Peptide transport system secreted peptide binding protein	7
12	MTD103	Homo sapiens	TMBV19 domain Family member B	8
13	MTD132	Streptomyces coelicolor	NP 628377 P60-family secreted protein	4
14	MTD151	Streptomyces coelicolor	NP_630126 Secreted chitinase	8
15	MTD173	Streptomyces coelicolor	NP 624384 Secreted protein	4
16	MTD174	Streptomyces coelicolor	NP_733505 Large, multifunctional secreted protein	8
17	MTD181	Neisseria meningitidis Z2491	CAB84257.1 Putative secreted protein	4

[0161] Table 2 shows the Summarizes Reference Information.

[Table 2]

#	References
	Title	Journal	Year	Vol	Issue	Page
1	Inhibition of Nuclear Translocation of Transcription Factor NF-kB by a Synthetic peptide Containing a Cell Membrane-permeable Motif and Nuclear Localization Sequence	JOURNAL OF BIOLOGICAL CHEMISTRY	1995	270	24	14255
2	Epigenetic Regulation of Gene Structure and Function with a Cell-Permeable Cre Recombinase	NATURE BIOTECHNOLOGY	2001	19	10	929
3	Cell-Permeable NM23 Blocks the Maintenance and Progression of Established Pulmonary Metastasis	CANCER RESEARCH	2011	71	23	7216
4	Antitumor Activity of Cell-Permeable p18INK4c With Enhanced Membrane and Tissue Penetration	MOLECULAR THERAPY	2012	20	8	1540
5	Antitumor Activity of Cell-Permeable RUNX3 Protein in Gastric Cancer Cells	CLINICAL CANCER RESEARCH	2012	19	3	680
6	The Effect of Intracellular Protein Delivery on the Anti-Tumor Activity of Recombinant Human Endostatin	BIOMATERIALS	2013	34	26	6261
7	Partial Somatic to Stem Cell Transformations Induced By Cell-Permeable Reprogramming Factors	SCIENTIFIC REPORTS	2014	4	10	4361
8	Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals	PLOS ONE	2014	9	7	17

[0162] Table 3 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (A) which were Analyzed.

[Table 3]

#	Peptides	Sequence	Length	Molecular Weight	pI	Bending Potential	Rigidity/Flexibility (Instability Index : II)	Structural Feature (Aliphatic Index : AI)	Hydropathy (GRAVY)	Residue Structure	A/a Composition						Secondary Structure	Cargo	Ref.
											A	V	L	I	P	G
1	MTM	AAVALLPAVLLALLAP	16	1,515.9	5.6	Bending	45.5	220.0	2.4	Aliphatic Ring	6	2	6	0	2	0	Helix	p50	1
2	MTS	AAVLLPVLLAAP	12	1,147.4	5.6	Bending	57.3	211.7	2.3	"	4	2	4	0	2	0	No-Helix	CRE	2
3	MTD10	LGGAVVAAPVAAAVAP	16	1,333.5	5.5	Bending	47.9	140.6	1.8	"	7	4	1	0	2	2	Helix	Parkin	8
4	MTD13	LAAAALAVLPL	11	1,022.3	5.5	Bending	26.6	213.6	2.4	"	5	1	4	0	1	0	No-Helix	RUNX3	3
5	MTD47	AAAVPVLVAA	10	881.0	5.6	Bending	47.5	176.0	2.4	"	5	3	1	0	1	0	No-Helix	CMYC	4
6	MTD56	VLLAAALIA	9	854.1	5.5	No-Bending	8.9	250.0	3.0	"	4	1	3	1	0	0	Helix	ES	5
7	MTD73	PVLLLLA	7	737.9	6.0	No -Bending	36.1	278.6	2.8	"	1	1	4	0	1	0	Helix	ES	5
8	MTD77	AVALLILAV	9	882.1	5.6	No -Bending	30.3	271.1	3.3	"	3	2	3	1	0	0	Helix	NM23	6
9	MTD84	AVALVAVVAVA	11	982.2	5.6	NO -Bending	9.1	212.7	3.1	"	5	5	1	0	0	0	Helix	OCT4	4
10	MTD85	LLAAAAALLLA	11	1,010.2	5.5	No -Bending	9.1	231.8	2.7	"	6	0	5	0	0	0	No-Helix	RUNX3	7
11	MTD86	LLAAAAALLLA	11	1,010.2	5.5	NO -Bending	9.1	231.8	2.7	"	6	0	5	0	0	0	No-Helix	SOX2	7
12	MTD103	LALPVLLLA	9	922.2	5.5	Bending	51.7	271.1	2.8	"	2	1	5	0	1	0	Helix	p18	8
13	MTD132	AVVVPAIVLAAP	12	1,119.4	5.6	Bending	50.3	195.0	2.4	"	4	4	1	1	2	0	No-Helix	LIN28	4
14	MTD151	AAAPVAAVP	9	1,031.4	5.5	Bending	73.1	120.0	1.6	∼							No-Helix	Parkin	8
15	MTD173	AVIPILAVP	9	892.1	5.6	Bending	48.5	216.7	2.4	"	2	2	1	2	2	0	Helix	KLF4	4
16	MTD174	LILLLPAVALP	12	1,011.8	5.5	Bending	79.1	257.3	2.6	"							Helix	Parkin	8
17	MTD181	AVLLLPAAA	9	838.0	5.6	Bending	51.7	206.7	2.4	"	4	1	3	0	1	0	No-Helix	SOX2	4
		AVE	10.8 ±2.4	1,011 ±189.6	5.6 ±0.1	Proline Presence	40.1 ±21.9	217.9 ±43.6	2.5 ±0.4

[0163] Two peptide/protein analysis programs were used (ExPasy: SoSui: http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) to determine various indexes and structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed.

1-2. Characteristics of Analyzed Peptides: Length, Molecular Weight and pl Value

[0164] Average length, molecular weight and pl value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively (Table 4)
Table 4 shows the Summarizes Critical Factors (CFs) of Published Hydrophobic Cell-Penetrating Peptides (A) which were Analyzed.

[Table 4]

• Length: 10.8 ± 2.4

• Molecular Weight: 1,011 ± 189.6

• pl: 5.6 ± 0.1

• Bending Potential (BP): Proline presences In the middle and/or the end of peptides, or No Proline.

• Instability Index (II): 40.1 ± 21.9

• Residue Structure & Aliphatic Index (AI): 217.9 ± 43.6

• Hydropathy (GRAVY): 2.5 ± 0.4

• Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I).

• Secondary Structure: α-Helix is favored but not required.

1-3. Characteristics of Analyzed Peptides: Bending Potential - Proline Position (PP)

[0165] Bending potential (bending or no-bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain.

[0166] Eleven out of 17 were determined as 'Bending' peptide which means that proline is present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate the plasma membrane in a "bent" configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.

1-4. Characteristics of Analyzed Peptides: Rigidity/Flexibility - Instability Index (II)

[0167] Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9 to 79.1), but average value was 40.1±21.9 which suggested that the peptide should be somehow flexible, but not too much rigid or flexible (Table 3).

1-5. Characteristics of Analyzed Peptides: Structural Features - Structural Feature (Aliphatic Index: AI) and Hydropathy (Grand Average of Hydropathy: GRAVY)

[0168] Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic - that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 (MTD10 - Table 3) and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively. Their amino acid composition is also indicated in the Table 3.

1-6. Characteristics of Analyzed Peptides: Secondary Structure (Helicity)

[0169] As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a "bent" configuration with hydrophobic sequences having α-helical conformation. In addition, our analysis strongly indicated that bending potential was crucial for membrane penetration. Therefore, structural analysis of the peptides was conducted to determine whether the sequences were to form helix or not. Nine peptides were helix and eight were not (Table 3). It seems to suggest that helix structure may not be required.

1-7. Determination of Critical Factors (CFs)

[0170] In the 11 characteristics analyzed, the following 6 are selected namely "Critical Factors" for the development of new hydrophobic CPPs - advanced MTDs: amino acid length, bending potential (proline presence and location), rigidity/flexibility (instability index: II), structural feature (aliphatic index: AI), hydropathy (GRAVY) and amino acid composition/residue structure (hydrophobic and aliphatic A/a) (Tables 3 and Table 4).

2. Analysis of Selected Hydrophobic CPPs to Optimize 'Critical Factors'

[0171] Since the analyzed data of the 17 different hydrophobic CPPs (analysis A, Tables 3 and 4) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus- features, analysis B (Tables 5 and 6) and C (Tables 7 and 8) were also conducted to optimize the critical factors for better design of improved CPPs - aMTDs. Therefore, 17 hydrophobic CPPs have been grouped into two groups and analyzed the groups for their characteristics in relation to the cell permeable property. The critical factors have been optimized by comparing and contrasting the analytical data of the groups and determining the common homologous features that may be critical for the cell permeable property.

2-1. Selective Analysis (B) of Peptides Used to Biologically Active Cargo Protein for In Vivo

[0172] In analysis B, eight CPPs were used with each biologically active cargo in vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility (instability index: II) was 41±15, but removing one [MTD85: rigid, with minimal II (9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy (Tables 5 and 6).

[0173] Table 5 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (B): Selected CPPs That were Used to Each Cargo In Vivo.

[Table 5]

#	Peptides	Sequence	Length	Molecular Weight	pI	Bending Potential	Rigidity/Flexibility (Instability Index : II)	Structural Feature (Aliphatic Index : AI)	Hydropathy (GRAVY)	Residue Structure	A/a Composition						Secondary Structure	Cargo	Ref.
											A	V	L	I	P	G
1	MTM	AAVALLPAVLLALLAP	16	1,515.9	5.6	Bending	45.5	220.0	2.4	Aliphatic Ring	6	2	6	0	2	0	Helix	p50	1
2	MTS	AAVLLPVLLAAP	12	1,147.4	5.6	Bending	57.3	211.7	2.3	"	4	2	4	0	2	0	No-Helix	CRE	2
3	MTD10	LGGAVVAAPVAAAVAP	16	1,333.5	5.5	Bending	47.9	140.6	1.8	"	7	4	1	0	2	2	Helix	Parkin	8
4	MTD73	PVLLLLA	7	737.9	6.0	No -Bending	36.1	278.6	2.8	"	1	1	4	0	1	0	Helix	ES	6
5	MTD77	AVALLILAV	9	882.1	5.6	No -Bending	30.3	271.1	3.3	"	3	2	3	1	0	0	Helix	NM23	3
6	MTD85	LLAAAAALLLA	11	1,010.2	5.5	-Bending	9.1*	231.8	2.7	"	6	0	5	0	0	0	No-Helix	RUNX3	5
7	MTD103	LALPVLLLA	9	922.2	5.5	Bending	51.7	271.1	2.8	"	2	1	5	0	1	0	Helix	p18	4
8	MTD132	AVVVPAIVLAAP	12	1,119.4	5.6	Bending	50.3	195.0	2.4	"	4	4	1	1	2	0	No-Helix	LIN28	7
		AVE	11±3.2	1,083±252	5.6±0.1	Proline Presence	41 ± 15	227 ± 47	2.5 ± 0.4

*Removing the MTD85 increases II to 45.6 ± 9.3.

[0174] Table 6 shows the Summarized Critical Factors of Published Hydrophobic Cell-Penetrating Peptides (B).

[Table 6]

• Length: 11 ± 3.2

• Molecular Weight: 1,083 ± 252

• pl: 5.6 ± 0.1

• Bending Potential (BP): Proline presences in the middle and/or the end of peptides, or No Proline.

• Instability Index (II): 41.0 ± 15 (▪ Removing the MTD85 increases II to 45.6 ± 9.3)

• Residue Structure & Aliphatic Index (AI): 227 ± 47

• Hydropathy (GRAVY): 2.5 ± 0.4

• Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I).

• Secondary Structure: α-Helix is favored but not required.

2-2. Selective Analysis (C) of Peptides That Provided Bending Potential and Higher Flexibility

[0175] To optimize the 'Common Range and/or Consensus Feature of Critical Factor' for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the 'Critical Factors' determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs - protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.

[0176] Hydrophobic CPPs which did noTo optimize the 'Common Range and/or Consensus Feature of Critical Factor' for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the 'Critical Factors' determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs - protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.

[0177] Hydrophobic CPPs which did not have a bending potential, rigid or too much flexible sequences (too much low or too much high Instability Index), or too low or too high hydrophobic CPPs were unselected, but secondary structure was not considered because helix structure of sequence was not required.

[0178] In analysis C, eight selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed (Table 7 and 8). Common amino acid length is 12 (11.6±3.0). Proline is presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5 to 57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs - aMTDs.

[0179] Table 7 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (C): Selected CPPs that Provided Bending Potential and Higher Flexibility.

[Table 7]

#	Peptides	Sequence	Length	Molecular Weight	pI	Bending Potential	Rigidity/Flexibility (Instability Index : II)	Structural Feature (Aliphatic Index : AI)	Hydropathy (GRAVY)	Residue Structure	A/a Composition						Secondary Structure	Cargo	Ref.
											A	V	L	I	P	G
1	MTM	AAVALLPAVLLALLAP	16	1515.9	5.6	Bending	45.5	220.0	2.4	Aliphatic Ring	6	2	6	0	2	0	Helix	p50	1
2	MTS	AAVLLPVLLAAP	12	1147.4	5.6	Bending	57.3	211.7	2.3	"	4	2	4	0	2	0	No-Helix	CRE	2
3	MTD10	LGGAVVAPVAAAVAP	16	1333.5	5.5	Bending	47.9	140.6	1.8	"	7	4	1	0	2	2	Helix	Parkin	8
4	MTD47	AAAVPVLVAA	10	881.0	5.6	Bending	47.5	176.0	2.4	"	5	3	1	0	1	0	No-Helix	CMYC	4
5	MTD103	LALPVLLLA	9	922.2	5.5	Bending	51.7	271.1	2.8	"	2	1	5	0	1	0	Helix	p18	8
6	MTD132	AVVVPAIVLAAP	12	1119.4	5.6	Bending	50.3	195.0	2.4	"	4	4	1	1	2	0	No-Helix	LIN28	4
7	MTD173	AVIPILAVP	9	892.1	5.6	Bending	48.5	216.7	2.4	"	2	2	1	2	2	0	Helix	KLF4	4
8	MTD181	AVLLLPAAA	9	838.0	5.6	Bending	51.7	206.7	2.4	"	4	1	3	0	1	0	No-Helix	SOX2	4
		AVE	11.6±3.0	1081.2±244.6	5.6±0.1	Proline Presence	50.1 ±3.6	204.7±37.5	2.4±0.3

[0180] Table 8 shows the Summarized Critical Factors of Published Hydrophobic Cell-Penetrating Peptides (C).

[Table 8]

• Length: 11.6 ± 3.0

• Molecular Weight: 1,081.2 ± 224.6

• pl: 5.6 ± 0.1

• Bending Potential (BP): Proline presences in the middle and/or the end of peptides.

• Instability Index (II): 50.1 ± 3.6

• Residue Structure & Aliphatic Index (AI): 204.7 ± 37.5

• Hydropathy (GRAVY): 2.4 ± 0.3

• Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I).

• Secondary Structure: α-Helix is favored but not required.

3. New Design of Improved Hydrophobic CPPs - aMTDs Based on the Optimized Critical Factors

3-1. Determination of Common Sequence and/or Common Homologous Structure

[0181] As mentioned above, H-regions of signal sequence (HOURSS)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, sequence motif, and/or common-structural homologous feature. According to one embodiment of the present invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence- and structural-motif which satisfy newly determined 'Critical Factors' to have 'Common Function,' namely, to facilitate protein translocation across the membrane with similar mechanism to the analyzed reference CPPs. Based on the analysis A, B and C, the common homologous features have been analyzed to determine the critical factors that influence the cell-permeability. The range value of each critical factor has been determined to include the analyzed index of each critical factor from analysis A, B and C to design novel aMTDs (Table 9). These features have been confirmed experimentally with newly designed aMTDs in their cell-permeability.

[0182] Table 9 shows the Comparison The Range/Feature of Each Critical Factor Between The Value of Analyzed CPPs and The Value Determined for New Design of Novel aMTDs Sequences.

[Table 9]

Summarized Critical Factors of aMTD
Critical Factor	Selected CPPs	Newly Designed CPPs
	Range	Range
Bending Potential (Proline Position: PP)	Proline presences in the middle and/or at the end of peptides	Proline presences in the middle (5', 6', 7' or 8') and at the end of peptides
Rigidity / Flexibility (Instability Index: II)	45.5 - 57.3 (50.1 ± 3.6)	40 - 60
Structural Feature (Aliphatic Index: AI)	140.6 - 220.0 (204.7 ± 37.5)	180 - 220
Hydropathy (Grand Average of Hydropathy GRAVY)	1.8 - 2.8 (2.4 ± 0.3)	2.1 - 2.6
Length (Number of Amino Acid)	11.6 ± 3.0	9 - 13
Amino acid Composition	A, V, I, L, P	A, V, I, L, P

[0183] In Table 9, universal common features and sequence/structural motif are provided. Length is 9 to 13 amino acids, and bending potential is provided with the presence of proline in the middle of sequence (at 5', 6', 7' or 8' amino acid) for peptide bending and at the end of peptide for recombinant protein bending and Rigidity/Flexibility of aMTDs is II > 40 are described in Table 9.

3-2. Critical Factors for Development of advanced MTDs

[0184] Recombinant cell-permeable proteins fused to the hydrophobic CPPs to deliver therapeutically active cargo molecules including proteins into live cells had previously been reported, but the fusion proteins expressed in bacteria system were hard to be purified as a soluble form due to their low solubility and yield. To address the crucial weakness for further clinical development of the cell-permeable proteins as protein-based biotherapeutics, greatly improved form of the hydrophobic CPP, named as advanced MTD (aMTD) has newly been developed through critical factors-based peptide analysis. The critical factors used for the current invention of the aMTDs are herein (Table 9).

1. Amino Acid Length: 9 to 13

2. Bending Potential (Proline Position: PP)

: Proline presences in the middle (from 5' to 8' amino acid) and at the end of sequence

3. Rigidity/Flexibility (Instability Index: II): 40 to 60

4. Structural Feature (Aliphatic Index: AI): 180 to 220

5. Hydropathy (GRAVY): 2.1 to 2.6

6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids to A, V, L, I and P

3-3. Design of Potentially Best aMTDs That All Critical Factors Are Considered and Satisfied

[0185] After careful consideration of six critical factors derived from analysis of unique features of hydrophobic CPPs, advanced macromolecule transduction domains (aMTDs) have been designed and developed based on the common 12 amino acid platform which satisfies the critical factors including amino acid length (9 to 13) determined from the analysis.

[0186] Unlike previously published hydrophobic CPPs that require numerous experiments to determine their cell-permeability, newly developed aMTD sequences could be designed by performing just few steps as follows using above mentioned platform to follow the determined range value/feature of each critical factor.

[0187] First, prepare the 12 amino acid sequence platform for aMTD. Second, place proline (P) in the end (12') of sequence and determine where to place proline in one of four U(s) in 5', 6', 7', and 8. Third, alanine (A), valine (V), leucine (L) or isoleucine (I) is placed in either X(s) and/or U(s), where proline is not placed. Lastly, determine whether the amino acid sequences designed based on the platform, satisfy the value or feature of six critical factors to assure the cell permeable property of aMTD sequences. Through these processes, numerous novel aMTD sequences have been constructed. The expression vectors for preparing non-functional cargo recombinant proteins fused to each aMTD, expression vectors have been constructed and forcedly expressed in bacterial cells. These aMTD-fused recombinant proteins have been purified in soluble form and determined their cell-permeability quantitatively. aMTD sequences have been newly designed, numbered from 1 to 240, as shown in Tables 10 to 15. In Tables 10 to 15, sequence ID Number is a sequence listings for reference, and aMTD numbers refer to amino acid listing numbers that actually have been used at the experiments. For further experiments, aMTD numbers have been used. In addition, polynucleotide sequences shown in the sequence lists have been numbered from SEQ ID NO : 241 to SEQ ID NO : 480.

[0188] Tables 10 to 15 show the 240 new hydrophobic aMTD sequences that were developed to satisfy all critical factors.

[Table 10]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
1	1	AAALAPVVLALP	12	57.3	187.5	2.1	Aliphatic
2	2	AAAVPLLAVVVP	12	41.3	195.0	2.4	Aliphatic
3	3	AALLVPAAVLAP	12	57.3	187.5	2.1	Aliphatic
4	4	ALALLPVAALAP	12	57.3	195.8	2.1	Aliphatic
5	5	AAALLPVALVAP	12	57.3	187.5	2.1	Aliphatic
6	11	VVALAPALAALP	12	57.3	187.5	2.1	Aliphatic
7	12	LLAAVPAVLLAP	12	57.3	211.7	2.3	Aliphatic
8	13	AAALVPVVALLP	12	57.3	203.3	2.3	Aliphatic
9	21	AVALLPALLAVP	12	57.3	211.7	2.3	Aliphatic
10	22	AVVLVPVLAAAP	12	57.3	195.0	2.4	Aliphatic
11	23	VVLVLPAAAAVP	12	57.3	195.0	2.4	Aliphatic
12	24	IALAAPALIVAP	12	50.2	195.8	2.2	Aliphatic
13	25	IVAVAPALVALP	12	50.2	203.3	2.4	Aliphatic
14	42	VAALPVVAVVAP	12	57.3	186.7	2.4	Aliphatic
15	43	LLAAPLVVAAVP	12	41.3	187.5	2.1	Aliphatic
16	44	ALAVPVALLVAP	12	57.3	203.3	2.3	Aliphatic
17	61	VAALPVLLAALP	12	57.3	211.7	2.3	Aliphatic
18	62	VALLAPVALAVP	12	57.3	203.3	2.3	Aliphatic
19	63	AALLVPALVAVP	12	57.3	203.3	2.3	Aliphatic

[Table 11]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
20	64	AIVALPVAVLAP	12	50.2	203.3	2.4	Aliphatic
21	65	IAIVAPVVALAP	12	50.2	203.3	2.4	Aliphatic
22	81	AALLPALAALLP	12	57.3	204.2	2.1	Aliphatic
23	82	AVVLAPVAAVLP	12	57.3	195.0	2.4	Aliphatic
24	83	LAVAAPLALALP	12	41.3	195.8	2.1	Aliphatic
25	84	AAVAAPLLLALP	12	41.3	195.8	2.1	Aliphatic
26	85	LLVLPAAALAAP	12	57.3	195.8	2.1	Aliphatic
27	101	LVALAPVAAVLP	12	57.3	203.3	2.3	Aliphatic
28	102	LALAPAALALLP	12	57.3	204.2	2.1	Aliphatic
29	103	ALIAAPILALAP	12	57.3	204.2	2.2	Aliphatic
30	104	AVVAAPLVLALP	12	41.3	203.3	2.3	Aliphatic
31	105	LLALAPAALLAP	12	57.3	204.1	2.1	Aliphatic
32	121	AIVALPALALAP	12	50.2	195.8	2.2	Aliphatic
33	123	AAIIVPAALLAP	12	50.2	195.8	2.2	Aliphatic
34	124	IAVALPALIAAP	12	50.3	195.8	2.2	Aliphatic
35	141	AVIVLPALAVAP	12	50.2	203.3	2.4	Aliphatic
36	143	AVLAVPAVLVAP	12	57.3	195.0	2.4	Aliphatic
37	144	VLAIVPAVALAP	12	50.2	203.3	2.4	Aliphatic
38	145	LLAVVPAVALAP	12	57.3	203.3	2.3	Aliphatic
39	161	AVIALPALIAAP	12	57.3	195.8	2.2	Aliphatic
40	162	AVVALPAALIVP	12	50.2	203.3	2.4	Aliphatic
41	163	LALVLPAALAAP	12	57.3	195.8	2.1	Aliphatic
42	164	LAAVLPALLAAP	12	57.3	195.8	2.1	Aliphatic
43	165	ALAVPVALAIVP	12	50.2	203.3	2.4	Aliphatic
44	182	ALIAPVVALVAP	12	57.3	203.3	2.4	Aliphatic
45	183	LLAAPVVIALAP	12	57.3	211.6	2.4	Aliphatic
46	184	LAAIVPAIIAVP	12	50.2	211.6	2.4	Aliphatic
47	185	AALVLPLIIAAP	12	41.3	220.0	2.4	Aliphatic
48	201	LALAVPALAALP	12	57.3	195.8	2.1	Aliphatic
49	204	LIAALPAVAALP	12	57.3	195.8	2.2	Aliphatic
50	205	ALALVPAIAALP	12	57.3	195.8	2.2	Aliphatic
51	221	AAILAPIVALAP	12	50.2	195.8	2.2	Aliphatic
52	222	ALLIAPAAVIAP	12	57.3	195.8	2.2	Aliphatic
53	223	AILAVPIAVVAP	12	57.3	203.3	2.4	Aliphatic
54	224	ILAAVPIALAAP	12	57.3	195.8	2.2	Aliphatic
55	225	VAALLPAAAVLP	12	57.3	187.5	2.1	Aliphatic
56	241	AAAVVPVLLVAP	12	57.3	195.0	2.4	Aliphatic
57	242	AALLVPALVAAP	12	57.3	187.5	2.1	Aliphatic
58	243	AAVLLPVALAAP	12	57.3	187.5	2.1	Aliphatic
59	245	AAALAPVLALVP	12	57.3	187.5	2.1	Aliphatic
60	261	LVLVPLLAAAAP	12	41.3	211.6	2.3	Aliphatic
61	262	ALIAVPAIIVAP	12	50.2	211.6	2.4	Aliphatic
62	263	ALAVIPAAAILP	12	54.9	195.8	2.2	Aliphatic
63	264	LAAAPVVIVIAP	12	50.2	203.3	2.4	Aliphatic
64	265	VLAIAPLLAAVP	12	41.3	211.6	2.3	Aliphatic
65	281	ALIVLPAAVAVP	12	50.2	203.3	2.4	Aliphatic
66	282	VLAVAPALIVAP	12	50.2	203.3	2.4	Aliphatic
67	283	AALLAPALIVAP	12	50.2	195.8	2.2	Aliphatic
68	284	ALIAPAVALIVP	12	50.2	211.7	2.4	Aliphatic
69	285	AIVLLPAAVVAP	12	50.2	203.3	2.4	Aliphatic

[Table 12]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
70	301	VIAAPVLAVLAP	12	57.3	203.3	2.4	Aliphatic
71	302	LALAPALALLAP	12	57.3	204.2	2.1	Aliphatic
72	304	AIILAPIAAIAP	12	57.3	204.2	2.3	Aliphatic
73	305	IALAAPILLAAP	12	57.3	204.2	2.2	Aliphatic
74	321	IVAVALPALAVP	12	50.2	203.3	2.3	Aliphatic
75	322	VVAIVLPALAAP	12	50.2	203.3	2.3	Aliphatic
76	323	IVAVALPVALAP	12	50.2	203.3	2.3	Aliphatic
77	324	IVAVALPAALVP	12	50.2	203.3	2.3	Aliphatic
78	325	IVAVALPAVALP	12	50.2	203.3	2.3	Aliphatic
79	341	IVAVALPAVLAP	12	50.2	203.3	2.3	Aliphatic
80	342	VIVALAPAVLAP	12	50.2	203.3	2.3	Aliphatic
81	343	IVAVALPALVAP	12	50.2	203.3	2.3	Aliphatic
82	345	ALLIVAPVAVAP	12	50.2	203.3	2.3	Aliphatic
83	361	AVVIVAPAVIAP	12	50.2	195.0	2.4	Aliphatic
84	363	AVLAVAPALIVP	12	50.2	203.3	2.3	Aliphatic
85	364	LVAAVAPALIVP	12	50.2	203.3	2.3	Aliphatic
86	365	AVIVVAPALLAP	12	50.2	203.3	2.3	Aliphatic
87	381	VVAIVLPAVAAP	12	50.2	195.0	2.4	Aliphatic
88	382	AAALVIPAILAP	12	54.9	195.8	2.2	Aliphatic
89	383	VIVALAPALLAP	12	50.2	211.6	2.3	Aliphatic
90	384	VIVAIAPALLAP	12	50.2	211.6	24	Aliphatic
91	385	IVAIAVPALVAP	12	50.2	203.3	2.4	Aliphatic
92	401	AALAVIPAAILP	12	54.9	195.8	2.2	Aliphatic
93	402	ALAAVIPAAILP	12	54.9	195.8	2.2	Aliphatic
94	403	AAALVIPAAILP	12	54.9	195.8	2.2	Aliphatic
95	404	LAAAVIPAAILP	12	54.9	195.8	2.2	Aliphatic
96	405	LAAAVIPVAILP	12	54.9	211.7	2.4	Aliphatic
97	421	AAILAAPLIAVP	12	57.3	195.8	2.2	Aliphatic
98	422	VVAILAPLLAAP	12	57.3	211.7	2.4	Aliphatic
99	424	AVVVAAPVLALP	12	57.3	195.0	2.4	Aliphatic
100	425	AVVAIAPVLALP	12	57.3	203.3	2.4	Aliphatic
101	442	ALAALVPAVLVP	12	57.3	203.3	2.3	Aliphatic
102	443	ALAALVPVALVP	12	57.3	203.3	2.3	Aliphatic
103	444	LAAALVPVALVP	12	57.3	203.3	2.3	Aliphatic
104	445	ALAALVPALVVP	12	57.3	203.3	2.3	Aliphatic
105	461	IAAVIVPAVALP	12	50.2	203.3	2.4	Aliphatic
106	462	IAAVLVPAVALP	12	57.3	203.3	2.4	Aliphatic
107	463	AVAILVPLLAAP	12	57.3	211.7	2.4	Aliphatic
108	464	AVVILVPLAAAP	12	57.3	203.3	2.4	Aliphatic
109	465	IAAVIVPVAALP	12	50.2	203.3	2.4	Aliphatic
110	481	AIAIAIVPVALP	12	50.2	211.6	2.4	Aliphatic
111	482	ILAVAAIPVAVP	12	54.9	203.3	2.4	Aliphatic
112	483	ILAAAIIPAALP	12	54.9	204.1	2.2	Aliphatic
113	484	LAVVLAAPAIVP	12	50.2	203.3	2.4	Aliphatic
114	485	AILAAIVPLAVP	12	50.2	211.6	2.4	Aliphatic
115	501	VIVALAVPALAP	12	50.2	203.3	2.4	Aliphatic
116	502	AIVALAVPVLAP	12	50.2	203.3	2.4	Aliphatic
117	503	AAIIIVLPAALP	12	50.2	220.0	2.4	Aliphatic
118	504	LIVALAVPALAP	12	50.2	211.7	2.4	Aliphatic
119	505	IAIIIVIAPAAAP	12	50.2	195.8	2.3	Aliphatic

[Table 13]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
120	521	LAALIVVPAVAP	12	50.2	203.3	2.4	Aliphatic
121	522	ALLVIAVPAVAP	12	57.3	203.3	2.4	Aliphatic
122	524	AVALIVVPALAP	12	50.2	203.3	2.4	Aliphatic
123	525	ALAIVVAPVAVP	12	50.2	195.0	2.4	Aliphatic
124	541	LLALIIAPAAAP	12	57.3	204.1	2.1	Aliphatic
125	542	ALALIIVPAVAP	12	50.2	211.6	2.4	Aliphatic
126	543	LLAALIAPAALP	12	57.3	204.1	2.1	Aliphatic
127	544	IVALIVAPAAVP	12	43.1	203.3	2.4	Aliphatic
128	545	VVLVLAAPAAVP	12	57.3	195.0	2.3	Aliphatic
129	561	AAVAIVLPAVVP	12	50.2	195.0	2.4	Aliphatic
130	562	ALIAAIVPALVP	12	50.2	211.7	2.4	Aliphatic
131	563	ALAVIVVPALAP	12	50.2	203.3	2.4	Aliphatic
132	564	VAIALIVPALAP	12	50.2	211.7	2.4	Aliphatic
133	565	VAIVLVAPAVAP	12	50.2	195.0	2.4	Aliphatic
134	582	VAVALIVPALAP	12	50.2	203.3	2.4	Aliphatic
135	583	AVILALAPIVAP	12	50.2	211.6	2.4	Aliphatic
136	585	ALIVAIAPALVP	12	50.2	211.6	2.4	Aliphatic
137	601	AAILIAVPIAAP	12	57.3	195.8	2.3	Aliphatic
138	602	VIVALAAPVLAP	12	50.2	203.3	2.4	Aliphatic
139	603	VLVALAAPVIAP	12	57.3	203.3	2.4	Aliphatic
140	604	VALIAVAPAVVP	12	57.3	195.0	2.4	Aliphatic
141	605	VIAAVLAPVAVP	12	57.3	195.0	2.4	Aliphatic
142	622	ALIVLAAPVAVP	12	50.2	203.3	2.4	Aliphatic
143	623	VAAAIALPAIVP	12	50.2	187.5	2.3	Aliphatic
144	625	ILAAAAAPLIVP	12	50.2	195.8	2.2	Aliphatic
145	643	LALVLAAPAIVP	12	50.2	211.6	2.4	Aliphatic
146	645	ALAVVALPAIVP	12	50.2	203.3	2.4	Aliphatic
147	661	AAILAPIVAALP	12	50.2	195.8	2.2	Aliphatic
148	664	ILIAIAIPAAAP	12	54.9	204.1	2.3	Aliphatic
149	665	LAIVLAAPVAVP	12	50.2	203.3	2.3	Aliphatic
150	666	AAIAIIAPAIVP	12	50.2	195.8	2.3	Aliphatic
151	667	LAVAIVAPALVP	12	50.2	203.3	2.3	Aliphatic
152	683	LAIVLAAPAVLP	12	50.2	211.7	2.4	Aliphatic
153	684	AAIVLALPAVLP	12	50.2	211.7	2.4	Aliphatic
154	685	ALLVAVLPAALP	12	57.3	211.7	2.3	Aliphatic
155	686	AALVAVLPVALP	12	57.3	203.3	2.3	Aliphatic
156	687	AILAVALPLLAP	12	57.3	220.0	2.3	Aliphatic
157	703	IVAVALVPALAP	12	50.2	203.3	2.4	Aliphatic
158	705	IVAVALLPALAP	12	50.2	211.7	2.4	Aliphatic
159	706	IVAVALLPAVAP	12	50.2	203.3	2.4	Aliphatic
160	707	IVALAVLPAVAP	12	50.2	203.3	2.4	Aliphatic
161	724	VAVLAVLPALAP	12	57.3	203.3	2.3	Aliphatic
162	725	IAVLAVAPAVLP	12	57.3	203.3	2.3	Aliphatic
163	726	LAVAIIAPAVAP	12	57.3	187.5	2.2	Aliphatic
164	727	VALAIALPAVLP	12	57.3	211.6	2.3	Aliphatic
165	743	AIAIALVPVALP	12	57.3	211.6	2.4	Aliphatic
166	744	AAVVIVAPVALP	12	50.2	195.0	2.4	Aliphatic
167	746	VAIIVVAPALAP	12	50.2	203.3	2.4	Aliphatic
168	747	VALLAIAPALAP	12	57.3	195.8	2.2	Aliphatic
169	763	IVAVLIAVPALAP	12	57.3	203.3	2.3	Aliphatic

[Table 14]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
170	764	AVALAVLPAVVP	12	57.3	195.0	2.3	Aliphatic
171	765	AVALAVVPAVLP	12	57.3	195.0	2.3	Aliphatic
172	766	IVVIAVAPAVAP	12	50.2	195.0	2.4	Aliphatic
173	767	IVVAAVVPALAP	12	50.2	195.0	2.4	Aliphatic
174	783	IVALVPAVAIAP	12	50.2	203.3	2.5	Aliphatic
175	784	VAALPAVALVVP	12	57.3	195.0	2.4	Aliphatic
176	786	LVAIAPLAVLAP	12	41.3	211.7	2.4	Aliphatic
177	787	AVALVPVIVAAP	12	50.2	195.0	2.4	Aliphatic
178	788	AIAVAIAPVALP	12	57.3	187.5	2.3	Aliphatic
179	803	AIALAVPVLALP	12	57.3	211.7	2.4	Aliphatic
180	805	LVLIAAAPIALP	12	41.3	220.0	2.4	Aliphatic
181	806	LVALAVPAAVLP	12	57.3	203.3	2.3	Aliphatic
182	807	AVALAVPALVLP	12	57.3	203.3	2.3	Aliphatic
183	808	LVVLAAAPLAVP	12	41.3	203.3	2.3	Aliphatic
184	809	LIVLAAPALAAP	12	502	195.8	2.2	Aliphatic
185	810	VIVLAAPALAAP	12	50.2	187.5	2.2	Aliphatic
186	811	AVVLAVPALAVP	12	57.3	195.0	2.3	Aliphatic
187	824	LIIVAAAPAVAP	12	50.2	187.5	2.3	Aliphatic
188	825	IVAVIVAPAVAP	12	43.2	195.0	2.5	Aliphatic
189	826	LVALAAPIIAVP	12	41.3	211.7	2.4	Aliphatic
190	827	IAAVLAAPALVP	12	57.3	187.5	2.2	Aliphatic
191	828	IALLAAPIIAVP	12	41.3	220.0	2.4	Aliphatic
192	829	AALALVAPVIVP	12	50.2	203.3	2.4	Aliphatic
193	830	IALVAAPVALVP	12	57.3	203.3	2.4	Aliphatic
194	831	IIVAVAPAAIVP	12	43.2	203.3	2.5	Aliphatic
195	832	AVAAIVPVIVAP	12	432	195.0	2.5	Aliphatic
196	843	AVLVLVAPAAAP	12	41.3	219.2	2.5	Aliphatic
197	844	VVALLAPLIAAP	12	41.3	211.8	2.4	Aliphatic
198	845	AAVVIAPLLAVP	12	41.3	203.3	2.4	Aliphatic
199	846	IAVAVAAPLLVP	12	41.3	203.3	2.4	Aliphatic
200	847	LVAIVVLPAVAP	12	50.2	219.2	2.6	Aliphatic
201	848	AVAIVVLPAVAP	12	50.2	195.0	2.4	Aliphatic
202	849	AVILLAPLIAAP	12	57.3	220.0	2.4	Aliphatic
203	850	LVIALAAPVALP	12	57.3	211.7	2.4	Aliphatic
204	851	VLAVVLPAVALP	12	57.3	219.2	2.5	Aliphatic
205	852	VLAVAAPAVLLP	12	57.3	203.3	2.3	Aliphatic
206	863	AAVVLLPIIAAP	12	41.3	211.7	2.4	Aliphatic
207	864	ALLVIAPAIAVP	12	57.3	211.7	2.4	Aliphatic
208	865	AVLVIAVPAIAP	12	57.3	203.3	2.5	Aliphatic
209	867	ALLVVIAPLAAP	12	41.3	211.7	2.4	Aliphatic
210	868	VLVAAILPAAIP	12	54.9	211.7	2.4	Aliphatic
211	870	VLVAAVLPIAAP	12	41.3	203.3	2.4	Aliphatic
212	872	VLAAAVLPLVVP	12	41.3	219.2	2.5	Aliphatic
213	875	AIAIVVPAVAVP	12	50.2	195.0	2.4	Aliphatic
214	877	VAIIAVPAVVAP	12	57.3	195.0	2.4	Aliphatic
215	878	IVALVAPAAVVP	12	50.2	195.0	2.4	Aliphatic
216	879	AAIVLLPAVVVP	12	50.2	219.1	2.5	Aliphatic
217	881	AALIVVPAVAVP	12	50.2	195.0	2.4	Aliphatic
218	882	AIALVVPAVAVP	12	57.3	195.0	2.4	Aliphatic
219	883	LAIVPAAIAALP	12	50.2	195.8	2.2	Aliphatic

[Table 15]

Sequence ID Number	aMTD	Sequences	Length	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Residue Structure
220	885	LVAIAPAVAVLP	12	57.3	203.3	2.4	Aliphatic
221	887	VLAVAPAVAVLP	12	57.3	195.0	2.4	Aliphatic
222	888	ILAVVAIPAAAP	12	54.9	187.5	2.3	Aliphatic
223	889	ILVAAAPIAALP	12	57.3	195.8	2.2	Aliphatic
224	891	ILAVAAIPAALP	12	54.9	195.8	2.2	Aliphatic
225	893	VIAIPAILAAAP	12	54.9	195.8	2.3	Aliphatic
226	895	AIIIVVPAIAAP	12	50.2	211.7	2.5	Aliphatic
227	896	AILIVVAPIAAP	12	50.2	211.7	2.5	Aliphatic
228	897	AVIVPVAIIAAP	12	50.2	203.3	2.5	Aliphatic
229	899	AVVIALPAVVAP	12	57.3	195.0	2.4	Aliphatic
230	900	ALVAVIAPVVAP	12	57.3	195.0	2.4	Aliphatic
231	901	ALVAVLPAVAVP	12	57.3	195.0	2.4	Aliphatic
232	902	ALVAPLLAVAVP	12	41.3	203.3	2.3	Aliphatic
233	904	AVLAVVAPVVAP	12	57.3	186.7	2.4	Aliphatic
234	905	AVIAVAPLVVAP	12	41.3	195.0	2.4	Aliphatic
235	906	AVIALAPVVVAP	12	57.3	195.0	2.4	Aliphatic
236	907	VAIALAPVVVAP	12	57.3	195.0	2.4	Aliphatic
237	908	VALALAPVVVAP	12	57.3	195.0	2.3	Aliphatic
238	910	VAALLPAVVVAP	12	57.3	195.0	2.3	Aliphatic
239	911	VALALPAVVVAP	12	57.3	195.0	2.3	Aliphatic
240	912	VALLAPAVVVAP	12	57.3	195.0	2.3	Aliphatic
				52.6 ± 5.1	201.7 ± 7.8	2.3 ± 0.1

3-4. Design of the Peptides That Did Not Satisfy at Least One Critical Factor

[0189] To demonstrate that one embodiment of the present invention of new hydrophobic CPPs - aMTDs, which satisfy all critical factors described above, are correct and rationally designed, the peptides which do not satisfy at least one critical factor have also been designed. Total of 31 rPeptides (rPs) are designed, developed and categorized as follows: no bending peptides, either no proline in the middle as well at the end and/or no central proline; rigid peptides (II < 40); too much flexible peptides; aromatic peptides (aromatic ring presences); hydrophobic, with non-aromatic peptides but have amino acids other than A, V, L, I, P or additional proline residues; hydrophilic, but non-aliphatic peptides.

3-4-1. Peptides That Do Not Satisfy the Bending Potential

[0190] Table 16 shows the peptides that do not have any proline in the middle (at 5', 6', 7' or 8') and at the end of the sequences. In addition, Table 16 describes the peptides that do not have proline in the middle of the sequences. All these peptides are supposed to have no-bending potential.

[Table 16]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)
No-Bending Peptides (No Proline at 5. 6. 7 or 8 and/or 12)	931	AVLIAPAILAAA	12	6	57.3	204.2	2.5
	936	ALLILAAAVAAP	12	12	41.3	204.2	2.4
	152	LAAAVAAVAALL	12	None	9.2	204.2	2.7
	27	LAIVAAAAALVA	12	None	2.1	204.2	2.8
	935	ALLILPAAAVAA	12	6	57.3	204.2	2.4
	670	ALLILAAAVAAL	12	None	25.2	236.6	2.8
	934	LILAPAAVVAA	12	5	57.3	195.8	2.5
	37	TTCSQQQYCTNG	12	None	53.1	0.0	-1.1
	16	NNSCTTYTNGSQ	12	None	47.4	0.0	-1.4
	113	PVAVALLIAVPP	12	1,11,12	57.3	195.0	2.1

3-4-2. Peptides That Do Not Satisfy the Rigidity/Flexibility

[0191] To prove that rigidity/flexibility of the sequence is a crucial critical factor, rigid (Avg. II: 21.8±6.6) and too high flexible sequences (Avg. II: 82.3±21.0) were also designed. Rigid peptides that instability index is much lower than that of new aMTDs (II: 41.3 to 57.3, Avg. II: 53.3±5.7) are shown in Table 17. Bending, but too high flexible peptides that II is much higher than that of new aMTDs are also provided in Table 18.

[Table 17]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)
	226	ALVAAIPALAIP	12	6	20.4	195.8	2.2
	6	VIAMIPAAFWVA	12	6	15.7	146.7	2.2
	750	LAIAAIAPLAIP	12	8.12	22.8	204.2	2.2
	26	AAIALAAPLAIV	12	8	18.1	204.2	2.5
	527	LVLAAVAPIAIP	12	8.12	22.8	211.7	2.4
	466	IIAAAAPLAIIP	12	7,12	22.8	204.2	2.3
Rigid Peptides (II < 50)	167	VAIAIPAALAIP	12	6.12	20.4	195.8	2.3
	246	VVAVPLLVAFAA	12	5	25.2	195.0	2.7
	426	AAALAIPLAIIP	12	7,12	4.37	204.2	2.2
	606	AAAIAAIPIIIP	12	8.12	4.4	204.2	2.4
	66	AGVLGGPIMGVP	12	7.12	35.5	121.7	1.3
	24B	VAAIVPIAALVP	12	6.12	34.2	203.3	2.5
	227	LAAIVPIAAAVP	12	6.12	34.2	187.5	2.2
	17	GGCSAPQTTCSN	12	6	51.6	8.3	-0.5
	67	LDAEVPLADDVP	12	6.12	34.2	130.0	0.3

[Table 18]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GARVY)
	692	PAPLPPVVILAV	12	1.3.5,6	105.5	186.7	1.8
	69	PVAVLPPAALVP	12	1,6,7,12	89.4	162.5	1.6
	390	VPLLVPVVPVVP	12	2,6,9,12	105.4	210.0	2.2
	350	VPILVPVVPVVP	12	2.6,9,12	121.5	210.0	2.2
	331	VPVLVPLVPVVP	12	2,6,9,12	105.4	210.0	2.2
	9	VALVPAALILPP	12	5,11,12	89.4	203.3	2.1
	68	VAPVLPAAPLVP	12	3,6,9,12	105.5	162.5	1.6
	349	VPVLVPVVPVVP	12	2,6,9,12	121.5	201.6	2.2
Bending Peptides but Too High Flexibility	937	VPVLVPLPVPVV	12	2,6,8,10	121.5	210.0	2.2
	938	VPVLLPVVVPVP	12	2,6,10,12	121.5	210.0	2.2
	329	LPVLVPVVPVVP	12	2,6,9,12	121.5	210.0	2.2
	49	VVPAAPAVPVVP	12	3.6,9.12	121.5	145.8	1.7
	772	lPVAPVIPIIVP	12	2,5,8,12	79.9	210.8	2.1
	210	ALIALPALPALP	12	6,9.12	89.4	195.8	1.8
	28	AVPLLPLVPAVP	12	3.6,9.12	89.4	186.8	1.8
	693	AAPVLPVAVPIV	12	3,6,10	82.3	186.7	2.1
	169	VALVAPALILAP	12	6,12	73.4	211.7	2.4
	29	VLPPLPVLPVLP	12	3,4,6,9,12	121.5	202.5	1.7
	190	AAILAPAVIAPP	12	6,11,12	89.4	163.3	1.8

3-4-3. Peptides That Do Not Satisfy the Structural Features

[0192] New hydrophobic CPPs - aMTDs are consisted with only hydrophobic and aliphatic amino acids (A, V, L, I and P) with average ranges of the indexes - AI: 180 to 220 and GRAVY: 2.1 to 2.6 (Table 9). Based on the structural indexes, the peptides which contain an aromatic residue (W, F or Y) are shown in Table 19 and the peptides which are hydrophobic with non-aromatic sequences but have amino acids residue other than A, V, L, I, P or additional proline residues are designed (Table 20). Finally, hydrophilic and/or bending peptides which are consisted with non-aliphatic amino acids are shown in Table 21.

[Table 19]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)
Aromatic Peptides (Aromatic Ring Presences)	30	WFFAGPIMLIWP	12	6,12	9.2	105.8	1.4
	33	AAAILAPAFLAV	12	7	57.3	171.7	2.4
	131	WIIAPVWLAWIA	12	5	51.6	179.2	1.9
	922	WYVIPVLPLVVP	12	8.12	41.3	194.2	2.2
	71	FMWMWFPFMWYP	12	7,12	71.3	0.0	0.6
	921	IWWPVVLPLVVP	12	8.12	41.3	194.2	2.2

[Table 20]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GARVY)
Hydrophobic but Non Aromatic Peptides	436	VVMLVVPAVMLP	12	7,12	57.3	194.2	2.6
	138	PPAALLAILAVA	12	1,2	57.3	195.8	2.2
	77	PVALVLVALVAP	12	1,12	41.3	219.2	2.5
	577	MLMIALVPMIAV	12	8	18.9	195.0	2.7
	97	ALLAAPPALLAL	12	6,7	57.3	204.2	2.1
	214	ALIVAPALMALP	12	6,12	60.5	187.5	2.2
	59	AVLAAPWAALA	12	6	41.3	187.5	2.5
	54	LAVAAPPVVALL	12	6,7	57.3	203.3	2.3

[Table 21]

Group	rPeptide ID	Sequences	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)
Hydrophilic Peptides but Non Aliphatic	949	SGNSCOOCGNSS	12	None	41.7	0.0	-1.1
	39	CYNTSPCTGCCY	12	6	52.5	0.0	0.0
	19	YVSCCTYTNGSO	12	None	47.7	0.0	-1.0
	947	CYYNOOSNNNNO	12	None	59.6	0.0	-2.4
	139	TGSTNSPTCTST	12	7	53.4	0.0	-0.7
	18	NYCCTPTTNGOS	12	6	47.9	0.0	-0.9
	20	NYCNTCPTYGOS	12	7	47.4	0.0	-0.9
	635	GSTGGSOONNOY	12	None	31.9	0.0	-1.9
	40	TYNTSCTPGTCY	12	8	49.4	0.0	-0.6
	57	ONNCNTSSOGGG	12	None	52.4	0.0	-1.6
	159	CYSGSTSONOPP	12	11.12	51.0	0.0	-1.3
	700	GTSNTCOSNONS	12	None	19.1	0.0	-1.6
	38	YYNOSTCGGOCY	12	None	53.8	0.0	-1.0

3-5. Summary of Newly Designed Peptides

[0193] Total of 457 sequences have been designed based on the critical factors. Designed potentially best aMTDs (hydrophobic, flexible, bending, aliphatic and 12-A/a length peptides) that do satisfy all range/feature of critical factors are 316. Designed rPeptides that do not satisfy at least one of the critical factors are 141 that no bending peptide sequences are 26; rigid peptide (II<40) sequences are 23; too much flexible peptides are 24; aromatic peptides (aromatic ring presences) are 27; hydrophobic, but non-aromatic peptides are 23; and hydrophilic, but non-aliphatic peptides are 18.

4. Preparation of Recombinant Report Proteins Fused to aMTDs and rPeptides

[0194] Recombinant proteins fused to aMTDs and others [rPeptides, reference hydrophobic CPP sequences (MTM and MTD)] were expressed in a bacterial system, purified with single-step affinity chromatography and prepared as soluble proteins in physiological condition. These recombinant proteins have been tested for the ability of their cell-permeability by utilizing flow cytometry and laser scanning confocal microscopy.

4-1. Selection of Cargo Protein for Recombinant Proteins Fused to Peptide Sequences

[0195] For clinical/non-clinical application, aMTD-fused cargo materials would be biologically active molecules that could be one of the following: enzymes, transcription factors, toxic, antigenic peptides, antibodies and antibody fragments. Furthermore, biologically active molecules could be one of these following macromolecules: enzymes, hormones, carriers, immunoglobulin, membrane-bound proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, virus proteins, native proteins, glycoproteins, fragmented proteins, disulfide bonded proteins, recombinant proteins, chemically modified proteins and prions. In addition, these biologically active molecules could be one of the following: nucleic acid, coding nucleic acid sequence, mRNAs, antisense RNA molecule, carbohydrate, lipids and glycolipids.

[0196] According to these pre-required conditions, a non-functional cargo to evaluate aMTD-mediated protein uptake has been selected and called as Cargo A (CRA) that should be soluble and non-functional. The domain (A/a 289 to 840; 184 A/a length) is derived from protein S (Genbank ID: CP000113.1).

4-2. Construction of Expression Vector and Preparation of Recombinant Proteins

[0197] Coding sequences for recombinant proteins fused to each aMTD are cloned Ndel (5') and SalI (3') in pET-28a(+) (Novagen, Darmstadt, Germany) from PCR-amplified DNA segments. PCR primers for the recombinant proteins fused to aMTD and rPeptides are represented by SEQ ID NOs: 481 to 797. Structure of the recombinant proteins is displayed in FIG. 1.

[0198] The recombinant proteins were forcedly expressed in E. coli BL21 (DE3) cells grown to an OD₆₀₀ of 0.6 and induced for 2 hours with 0.7 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni²⁺ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany) in natural condition. After the purification, purified proteins were dissolved in a physiological buffer such as DMEM medium.

[Table 22]

► Potentially Best aMTDs (Hydrophobic, Flexible, Bending, Aliphatic & Helical)	: 240
► Random Peptides	: 31
- No Bending Peptides (No Proline at 5 or 6 and/or 12)	: 02
- No Bending Peptides (No Central Proline)	: 01
- Rigid Peptides (II<50)	: 09
- Too Much Flexible Peptides	: 09
- Aromatic Peptides (Aromatic Ring Presences)	: 01
- Hydrophobic, But Non-Aromatic Peptides	: 02
- Hydrophilic, But Non-Aliphatic Peptides	: 07

4-3. Expression of aMTD- or Random Peptide (rP)- Fused Recombinant Proteins

[0199] One embodiment of the present invention also relates to the development method of aMTD sequences having cell-permeability. Using the standardized six critical factors, 316 aMTD sequences have been designed. In addition, 141 rPeptides are also developed that lack one of these critical factors: no bending peptides: i) absence of proline both in the middle and at the end of sequence or ii) absence of proline either in the middle or at the end of sequence, rigid peptides, too much flexible peptides, aromatic peptides (aromatic ring presence), hydrophobic but non-aromatic peptides, and hydrophilic but non-aliphatic peptides (Table 22).

[0200] These rPeptides are devised to be compared and contrasted with aMTDs in order to analyze structure/sequence activity relationship (SAR) of each critical factor with regard to the peptides' intracellular delivery potential. All peptide (aMTD or rPeptide)-containing recombinant proteins have been fused to the CRA to enhance the solubility of the recombinant proteins to be expressed, purified, prepared and analyzed.

[0201] These designed 316 aMTDs and 141 rPeptides fused to CRA were all cloned (FIG. 2) and tested for inducible expression in E.coli (FIG. 3). Out of these peptides, 240 aMTDs were inducibly expressed, purified and prepared in soluble form (FIG. 4). In addition, 31 rPeptides were also prepared as soluble form (FIG. 4).

[0202] To prepare the proteins fused to rPeptides, 60 proteins were expressed that were 10 out of 26 rPeptides in the category of no bending peptides (Table 16); 15 out of 23 in the category of rigid peptides [instability index (II) < 40 ] (Table 17); 19 out of 24 in the category of too much flexible peptides (Table 18); 6 out of 27 in the category of aromatic peptides (Table 19); 8 out of 23 in the category of hydrophobic but non-aromatic peptides (Table 20); and 12 out of 18 in the category of hydrophilic but non-aliphatic peptides (Table 21).

4-4. Quantitative Cell-Permeability of aMTD-Fused Recombinant Proteins

[0203] The aMTDs and rPeptides were fluorescently labeled and compared based on the critical factors for cell-permeability by using flow cytometry and confocal laser scanning microscopy (FIGs. 5 to 8). The cellular uptake of the peptide-fused non-functional cargo recombinant proteins could quantitatively be evaluated in flow cytometry, while confocal laser scanning microscopy allows intracellular uptake to be assessed visually. The analysis included recombinant proteins fused to a negative control [rP38] that has opposite characteristics (hydrophilic and aromatic sequence: YYNQSTCGGQCY) to the aMTDs (hydrophobic and aliphatic sequences). Relative cell-permeability (relative fold) of aMTDs to the negative control was also analyzed (Table 23 and FIG. 9).

[0204] Table 23 shows the Comparison Analysis of Cell-Permeability of aMTDs with a Negative Control (A: rP38).

[Table 23]

	Negative Control rP38
aMTD The Average of 240 aMTDs	19.6 ± 1.6* (Best: 164.2)

*Relative Fold (aMTD in Geo Mean in its comparison to rP38)

[0205] Relative cell-permeability (relative fold) of aMTDs to the reference CPPs [B: MTM12 (AAVLLPVLLAAP), C: MTD85 (AVALLILAV)] was also analyzed (Tables 40 and 41)
Table 24 shows Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (B: MTM12).

[Table 24]

	MTM12
aMTD The Average of 240 aMTDs	13.1 ± 1.1* (Best: 109.9)

*Relative Fold (aMTD in Geo Mean in its comparison to MTM12)

[0206] Table 25 shows the Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (C: MTD85).

[Table 25]

	MTD85
aMTD The Average of 240 aMTDs	6.6±0.5* (Best: 55.5)

*Relative Fold (aMTD in Geo Mean in its comparison to MTD85)

[0207] Geometric means of negative control (histidine-tagged rP38-fused CRA recombinant protein) subtracted by that of naked protein (histidine-tagged CRA protein) lacking any peptide (rP38 or aMTD) was standardized as relative fold of 1. Relative cell-permeability of 240 aMTDs to the negative control (A type) was significantly increased by up to 164 fold, with average increase of 19.6±1.6 (Tables 26 to 31).

[Table 26]

Sequence ID Number	aMTD	Sequences	Length	Proline Position	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)
				(PP)	(II)	(AI)	(GRAVY)	A	B	C
1	899	AVVIALPAVVAP	12	7	57.3	195.0	2.4	164.2	109.9	55.5
2	908	VALALAPVVVAP	12	7	57.3	195.0	2.3	150.6	100.8	50.9
3	910	VAALLPAVVVAP	12	6	57.3	195.0	2.3	148.5	99.4	50.2
4	810	VIVLAAPALAAP	12	7	50.2	187 .5	2.2	120.0	80.3	40.6
5	904	AVLAVVAPVVAP	12	8	57.3	186.7	2.4	105.7	70.8	35.8
6	321	IVAVALPALAVP	12	7	50.2	203.3	2.3	97.8	65.2	32.9
7	851	VLAVVLPAVALP	12	7	57.3	219.2	2.5	96.6	64.7	32.7
8	911	VALALPAVVVAP	12	6	57.3	195.0	2.3	84.8	56.8	28.7
9	852	VLAVAAPAVLLP	12	7	57.3	203.3	2.3	84.6	56.6	28.6
10	803	AIALAVPVLALP	12	7	57.3	211.7	24	74.7	50.0	25.3
11	888	ILAVVAIPAAAP	12	8	54.9	187.5	2.3	71.0	47.5	24.0
12	825	IVAVIVAPAVAP	12	8	43.2	195.0	2.5	69.7	46.6	23.6
13	895	AIIIVVPAIAAP	12	7	50.2	211.7	2.5	60.8	40.7	20.6
14	896	AILIVVAPIAAP	12	8	50.2	211.7	2.5	57.5	38.5	19.4
15	727	VALAIALPAVLP	12	8	57.3	211.6	2.3	54.7	36.7	18.5
16	603	VLVALAAPVIAP	12	8	57.3	203.3	2.4	54.1	36.1	18.2
17	847	LVAIVVLPAVAP	12	8	50.2	219.2	2.6	50.2	33.4	16.9
18	826	LVALAAPIIAVP	12	7	41.3	211.7	2.4	49.2	32.9	16.6
19	724	VAVLAVLPALAP	12	8	57.3	203.3	2.3	47.5	31.8	16.1
20	563	ALAVIVVPALAP	12	8	50.2	203.3	2.4	47.1	31.4	15.9
21	811	AVVLAVPALAVP	12	7	57.3	195.0	2.3	46.5	31.1	15.7
22	831	IIVAVAPAAIVP	12	7	43.2	203.3	2.5	46.3	31.0	15.7
23	829	AALALVAPVIVP	12	8	50.2	203.3	2.4	44.8	30.0	15.2
24	891	ILAVAAIPAALP	12	8	54.9	195.8	2.2	44.7	29.9	15.1
25	905	AVIAVAPLVVAP	12	7	41.3	195.0	2.4	44.0	295	14.9
26	564	VAIALIVPALAP	12	8	50.2	211.7	2.4	43.6	29.1	14.7
27	124	IAVALPALIAAP	12	6	50.3	195.8	2.2	43.6	29.0	14.7
28	827	IAAVLAAPALVP	12	8	57.3	187.5	2.2	43.0	28.8	14.6
29	2	AAAVPLLAVVVP	12	5	41.3	195.0	2.4	40.9	27.2	13.8
30	385	IVAIAVPALVAP	12	7	50.2	203.3	2.4	38.8	25.9	13.1
31	828	IALLAAPIIAVP	12	7	41.3	220.0	2.4	36.8	24.6	12.4
32	806	LVALAVPAAVLP	12	7	57.3	203.3	2.3	36.7	24.6	12.4
33	845	AAVVIAPLLAVP	12	7	41.3	203.3	2.4	35.8	24.0	12.1
34	882	AIALVVPAVAVP	12	7	57.3	195.0	2.4	35.0	23.4	11.8
35	545	VVLVLAAPAAVP	12	8	57.3	195.0	2.3	34.6	23.1	11.7
36	161	AVIALPALIAAP	12	6	57.3	195.8	2.2	34.5	23.0	11.6
37	481	AIAIAIVPVALP	12	8	50.2	211.6	2.4	34.3	23.0	11.6
38	900	ALVAVIAPVVAP	12	8	57.3	195.0	2.4	34.3	22.9	11.6
39	223	AILAVPIAVVAP	12	6	57.3	203.3	2.4	33.0	22.1	11.2
40	824	LIIVAAAPAVAP	12	8	50.2	187.5	2.3	32.8	21.9	11.1
41	562	ALIAAIVPALVP	12	8	50.2	211.7	2.4	32.7	21.8	11.0
42	222	ALLIAPAAVIAP	12	6	57.3	195.8	2.2	32.6	21.7	11.0
43	61	VAALPVLLAALP	12	5	57.3	211.7	2.3	31.2	20.8	10.5
44	582	VAVALIVP ALAP	12	8	50.2	203.3	2.4	30.6	20.4	10.3
45	889	ILVAAAPIAALP	12	7	57.3	195.8	2.2	30.3	20.3	10.3
46	787	AVALVPVIVAAP	12	6	50.2	195.0	2.4	29.3	19.6	9.9
47	703	IVAVALVPALAP	12	8	50.2	203.3	2.4	29.2	19.5	9.9
48	705	IVAVALLP ALAP	12	8	50.2	211.7	2.4	28.6	19.1	9.7
49	885	LVAIAPAVAVLP	12	6	57.3	203.3	2.4	28.3	19.0	9.6
50	3	AALLVPAAVLAP	12	6	57.3	187.5	2.1	27.0	18.0	9.1
51	601	AAILIAVPIAAP	12	8	57.3	195.8	2.3	26.8	17.9	9.0
52	843	AVLVLVAPAAAP	12	8	41.3	219.2	2.5	26.4	17.7	8.9
53	403	AAALVIPAAILP	12	7	54.9	195.8	2.2	25.2	16.8	8.5
54	544	IVALIVAPAAVP	12	8	43.1	203.3	2.4	23.4	15.6	7.9
55	522	ALLVIAVPAVAP	12	8	57.3	203.3	2.4	22.7	15.2	7.7

[Table 27]

Sequence ID Number	aMTD	Sequences	Length	Proline Position	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)
				(PP)	(II)	(AI)	(GRAVY)	A	B	C
56	805	LVLIAAAPIALP	12	8	41.3	220.0	2.4	22.3	14.9	7.6
57	464	AVVILVPLAAAP	12	7	57.3	203.3	2.4	22.3	14.9	7.5
58	405	LAAAVIPVAILP	12	7	549	211.7	2.4	22.2	14.8	7.5
59	747	VALLAIAPALAP	12	8	57.3	195.8	2.2	22.0	14.8	7.5
60	501	VIVALAVPALAP	12	8	50.2	203.3	2.4	21.5	14.4	7.3
61	661	AAILAPIVAALP	12	6	50.2	195.8	2.2	21.4	14.3	7.2
62	786	LVAIAPLAVLAP	12	6	41.3	211.7	2.4	21.2	14.2	7.2
63	625	ILAAAAAPLIVP	12	8	50.2	195.8	2.2	20.9	13.9	7.0
64	442	ALAALVPAVLVP	12	7	57.3	203.3	2.3	20.4	13.6	6.9
65	912	VALLAPAVVVAP	12	6	57.3	195.0	2.3	19.9	13.3	6.7
66	165	ALAVPVALAIVP	12	5	50.2	203.3	2.4	19.8	13.2	6.7
67	422	VVAILAPLLAAP	12	7	57.3	211.7	2.4	19.6	13.1	6.6
68	686	AALVAVLPVALP	12	8	57.3	203.3	2.3	19.5	13.1	6.6
69	343	IVAVALPALVAP	12	7	50.2	203.3	2.3	19.4	12.9	6.5
70	323	IVAVALPVALAP	12	7	50.2	203.3	2.3	19.1	12.8	6.4
71	461	IAAVIVPAVALP	12	7	50.2	203.3	2.4	19.0	12.7	6.4
72	21	AVALLPALLAVP	12	6	57.3	211.7	2.3	18.9	12.6	6.4
73	404	LAAAVIPAAILP	12	7	54.9	195.8	2.2	18.9	12.6	6.4
74	261	LVLVPLLAAAAP	12	5	41.3	211.6	2.3	18.5	12.3	6.2
75	524	AVALIVVPALAP	12	8	50.2	203.3	2.4	18.3	12.2	6.2
76	225	VAALLPAAAVLP	12	6	57.3	187.5	2.1	18.3	12.2	6.2
77	264	LAAAPVVIVIAP	12	5	50.2	203.3	2.4	18.2	12.1	6.1
78	1	AAALAPVVLALP	12	6	57.3	187.5	2.1	17.7	11.8	6.0
79	382	AAALVIPAILAP	12	7	54.9	195.8	2.2	17.7	11.8	6.0
80	463	AVAILVPLLAAP	12	7	57.3	211.7	2.4	17.6	11.7	5.9
81	322	VVAIVLPALAAP	12	7	50.2	203.3	2.3	17.6	11.7	5.9
82	503	AAIIIVLPAALP	12	8	50.2	220.0	2.4	17.6	11.8	5.9
83	870	VLVAAVLPIAAP	12	8	41.3	203.3	2.4	16.6	11.1	5.6
84	241	AAAWPVLLVAP	12	6	57.3	195.0	2.4	16.6	11.0	5.6
85	726	LAVAIIAP AVAP	12	8	57.3	187.5	2.2	16.5	11.0	5.6
86	341	IVAVALPAVLAP	12	7	50.2	203.3	2.3	16.4	10.9	5.5
87	542	ALALIIVPAVAP	12	8	50.2	211.6	2.4	16.2	10.8	5.5
88	361	AVVIVAPAVIAP	12	7	50.2	195.0	2.4	16.0	10.7	5.4
89	224	ILAAVPIALAAP	12	6	57.3	195.8	2.2	15.8	10.6	5.3
90	482	ILAVAAIPVAVP	12	8	54.9	203.3	2.4	15.8	10.6	5.3
91	64	AIVALPVAVLAP	12	6	50.2	203.3	2.4	15.8	10.6	5.3
92	484	LAVVLAAPAIVP	12	8	50.2	203.3	2.4	15.6	10.4	5.3
93	868	VLVAAILPAAIP	12	8	54.9	211.7	2.4	14.9	10.0	5.0
94	541	LLALIIAP AAAP	12	8	57.3	204.1	2.1	14.8	9.9	5.0
95	666	AAIAIIAPAIVP	12	8	50.2	195.8	2.3	14.7	9.9	5.0
96	665	LAIVLAAPVAVP	12	8	50.2	203.3	2.3	14.7	9.9	5.0
97	363	AVLAVAPALIVP	12	7	50.2	203.3	2.3	14.7	9.8	4.9
98	242	AALLVPALVAAP	12	6	57.3	187.5	2.1	14.6	9.7	4.9
99	384	VIVAIAPALLAP	12	7	50.2	211.6	2.4	14.0	9.4	4.7
100	877	VAIIAVPAVVAP	12	7	57.3	195.0	2.4	14.0	9.4	4.7
101	863	AAVVLLPIIAAP	12	7	41.3	211.7	2.4	13.8	9.3	4.7
102	525	ALAIVVAPVAVP	12	8	502	195.0	2.4	13.8	9.2	4.7
103	875	AIAIVVPAVAVP	12	7	50.2	195.0	2.4	13.8	9.2	4.7
104	285	AIVLLPAAVVAP	12	6	50.2	203.3	2.4	13.3	8.9	4.5
105	281	ALIVLPAAVAVP	12	6	50.2	203.3	2.4	13.3	8.9	4.5
106	867	ALLVVIAPLAAP	12	8	41.3	211.7	2.4	13.2	8.8	4.4
107	766	IVVIAVAPAVAP	12	8	50.2	195.0	2.4	12.9	8.6	4.4
108	342	VIVALAPAVLAP	12	7	50.2	203.3	2.3	12.7	8.5	4.3
109	881	AALIVVPAVAVP	12	7	50.2	195.0	2.4	12.7	8.5	4.3
110	505	AIIIVIAPAAAP	12	8	50.2	195.8	2.3	12.4	8.3	4.2

[Table 28]

Sequence ID Number	aMTD	Sequences	Length	Proline Position	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)
				(PP)	(II)	(AI)	(GRAVY)	A	B	C
111	763	VAVLIAVPALAP	12	8	57.3	203.3	2.3	12.3	7.2	4.2
112	706	IVAVALLPAVAP	12	8	50.2	203.3	2.4	12.0	7.0	4.1
113	687	AILAVALPLLAP	12	8	57.3	220.0	2.3	12.0	7.0	4.1
114	643	LALVLAAPAIVP	12	8	50.2	211.6	2.4	11.8	7.9	4.0
115	282	VLAVAPALIVAP	12	6	50.2	203.3	2.4	11.8	7.9	4.0
116	543	LLAALIAPAALP	12	8	57.3	204.1	2.1	11.7	7.8	4.0
117	325	IVAVALPAVALP	12	7	50.2	203.3	2.3	11.7	7.8	4.0
118	846	IAVAVAAPLLVP	12	8	41.3	203.3	2.4	11.7	6.8	4.0
119	383	VIVALAPALLAP	12	7	50.2	211.6	2.3	11.6	7.7	3.9
120	381	VVAIVLPAVAAP	12	7	50.2	195.0	2.4	11.5	7.7	3.9
121	808	LVVLAAAPLAVP	12	8	41.3	203.3	2.3	11.5	7.6	3.9
122	865	AVLVIAVPAIAP	12	8	57.3	203.3	2.5	11.3	7.5	3.8
123	725	IAVLAVAPAVLP	12	8	57.3	203.3	2.3	11.2	7.5	3.8
124	844	VVALLAPLIAAP	12	7	41.3	211.8	2.4	11.2	7.5	3.8
125	897	AVIVPVAIIAAP	12	5	50.2	203.3	2.5	11.2	7.5	3.8
126	605	VIAAVLAPVAVP	12	8	57.3	195.0	2.4	11.0	7.4	3.7
127	744	AAVVIVAPVALP	12	8	50.2	195.0	2.4	11.0	7.3	3.7
128	221	AAILAPIVALAP	12	6	50.2	195.8	2.2	10.9	7.3	3.7
129	622	ALIVLAAPVAVP	12	8	50.2	203.3	2.4	10.6	7.1	3.6
130	401	AALAVIPAAILP	12	7	54.9	195.8	2.2	10.6	7.1	3.6
131	324	IVAVALPAALVP	12	7	50.2	203.3	2.3	10.3	6.9	3.5
132	878	IVALVAPAAVVP	12	7	50.2	195.0	2.4	10.3	6.9	3.5
133	302	LALAPALALLAP	12	5	57.3	204.2	2.1	10.2	6.8	3.4
134	685	ALLVAVLPAALP	12	8	57.3	211.7	2.3	10.2	5.9	3.4
135	848	AVAIVVLPAVAP	12	8	50.2	195.0	2.4	10.0	6.7	3.4
136	602	VIVALAAPVLAP	12	8	50.2	203.3	2.4	9.9	5.8	3.4
137	788	AIAVAIAPVALP	12	8	57.3	187.5	2.3	9.8	6.6	3.3
138	145	LLAVVPAVALAP	12	6	57.3	203.3	2.3	9.5	6.3	3.2
139	11	VVALAPALAALP	12	6	57.3	187.5	2.1	9.5	6.3	3.2
140	141	AVIVLPALAVAP	12	6	50.2	203.3	2.4	9.4	6.3	3.2
141	521	LAALIVVPAVAP	12	8	50.2	203.3	2.4	9.4	6.3	3.2
142	425	AVVAIAPVLALP	12	7	57.3	203.3	2.4	9.4	6.3	3.2
143	365	AVIVVAPALLAP	12	7	50.2	203.3	2.3	9.3	6.2	3.1
144	263	ALAVIPAAAILP	12	6	54.9	195.8	2.2	9.0	6.0	3.0
145	345	ALLIVAPVAVAP	12	7	50.2	203.3	2.3	8.9	5.9	3.0
146	850	LVIALAAPVALP	12	8	57.3	211.7	2.4	8.8	5.9	3.0
147	144	VLAIVPAVALAP	12	6	50.2	203.3	2.4	8.8	5.9	3.0
148	767	IVVAAVVPALAP	12	8	50.2	195.0	2.4	8.5	5.0	2.9
149	185	AALVLPLIIAAP	12	6	41.3	220.0	2.4	8.5	5.7	2.9
150	849	AVILLAPLIAAP	12	7	57.3	220.0	2.4	8.3	4.8	2.8
151	864	ALLVIAPAIAVP	12	7	57.3	211.7	2.4	8.2	4.8	2.8
152	162	AVVALPAALIVP	12	6	50.2	203.3	2.4	8.2	5.5	2.8
153	164	LAAVLPALLAAP	12	6	57.3	195.8	2.1	8.2	5.5	2.8
154	907	VAIALAPVVVAP	12	7	57.3	195.0	2.4	8.1	5.4	2.8
155	444	LAAALVPVALVP	12	7	57.3	203.3	2.3	8.1	5.4	2.7
156	443	ALAALVPVALVP	12	7	57.3	203.3	2.3	8.0	5.3	2.7
157	901	ALVAVLPAVAVP	12	7	57.3	195.0	2.4	7.7	5.1	2.6
158	887	VLAVAPAVAVLP	12	6	57.3	195.0	2.4	7.7	5.1	2.6
159	746	VAIIVVAPALAP	12	8	50.2	203.3	2.4	7.6	4.4	2.6
160	902	ALVAPLLAVAVP	12	5	41.3	203.3	2.3	7.6	5.1	2.6
161	565	VAIVLVAPAVAP	12	8	50.2	195.0	2.4	7.5	5.0	2.5
162	245	AAALAPVLALVP	12	6	57.3	187.5	2.1	7.5	5.0	2.5
163	743	AIAIALVPVALP	12	8	57.3	211.6	2.4	7.4	4.9	2.5
164	465	AVVILVP LAAAP	12	7	57.3	203.3	24	7.4	4.9	2.5
165	104	AVVAAPLVLALP	12	6	41.3	203.3	2.3	7.3	4.9	2.5

[Table 29]

Sequence ID Number	aMTD	Sequences	Length	Proline Position	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)
				(PP)	(II)	(AI)	(GRAVY)	A	B	C
166	707	IVALAVLPAVAP	12	8	50.2	203.3	2.4	7.3	4.9	2.5
167	872	VLAAAVLPLVVP	12	8	41.3	219.2	2.5	7.3	4.9	2.5
168	583	AVILALAPIVAP	12	8	50.2	211.6	2.4	7.3	4.8	2.4
169	879	AAIVLLPAVVVP	12	7	50.2	219.1	2.5	7.2	4.8	2.4
170	784	VAALPAVALVVP	12	5	57.3	195.0	2.4	7.1	4.7	2.4
171	893	VIAIPAILAAAP	12	5	54.9	195.8	2.3	7.0	4.7	2.4
172	13	AAALVPVVALLP	12	6	57.3	203.3	2.3	7.0	4.7	2.4
173	809	LIVLAAPALAAP	12	7	50.2	195.8	2.2	7.0	4.7	2.4
174	445	ALAALVPALVVP	12	7	57.3	203.3	2.3	6.9	4.6	2.3
175	81	AALLPALAALLP	12	5	57.3	204.2	2.1	6.9	4.6	2.3
176	667	LAVAIVAPALVP	12	8	50.2	203.3	2.3	6.9	4.6	2.3
177	906	AVIALAPVVVAP	12	7	57.3	195.0	2.4	6.8	4.6	2.3
178	483	ILAAAIIPAALP	12	8	54.9	204.1	2.2	6.8	4.5	2.3
179	485	AILAAIVPLAVP	12	8	50.2	211.6	2.4	6.8	4.5	2.3
180	421	AAILAAPLIAVP	12	7	57.3	195.8	2.2	6.7	4.5	2.3
181	585	ALIVAIAPALVP	12	8	50.2	211.6	2.4	6.6	4.4	2.2
182	424	AVVVAAPVLALP	12	7	57.3	195.0	2.4	6.6	4.4	2.2
183	364	LVAAVAPALIVP	12	7	50.2	203.3	2.3	6.5	4.3	2.2
184	402	ALAAVIPAAILP	12	7	54.9	195.8	2.2	6.4	4.3	2.2
185	462	IAAVLVPAVALP	12	7	57.3	203.3	2.4	6.3	4.2	2.1
186	265	VLAIAPLLAAVP	12	6	41.3	211.6	2.3	6.0	4.0	2.0
187	301	VIAAPVLAVLAP	12	6	57.3	203.3	2.4	6.0	4.0	2.0
188	183	LLAAPVVIALAP	12	6	57.3	211.6	2.4	6.0	4.0	2.0
189	243	AAVLLPVALAAP	12	6	57.3	187.5	2.1	5.9	3.9	2.0
190	664	ILIAIAIPAAAP	12	8	54.9	204.1	2.3	5.7	3.8	1.9
191	783	IVALVPAVAIAP	12	6	50.2	203.3	2.5	5.7	3.8	1.9
192	502	AIVALAVPVLAP	12	8	50.2	203.3	2.4	5.6	3.7	1.9
193	262	ALIAVPAIIVAP	12	6	50.2	211.6	2.4	5.5	3.7	1.9
194	683	LAIVLAAPAVLP	12	8	50.2	211.7	2.4	5.5	3.2	1.9
195	830	IALVAAPVALVP	12	7	57.3	203.3	2.4	5.3	3.5	1.8
196	764	AVALAVLPAVVP	12	8	57.3	195.0	2.3	5.0	3.4	1.7
197	807	AVALAVPALVLP	12	7	57.3	203.3	2.3	5.0	3.3	1.7
198	184	LAAIVPAIIAVP	12	6	50.2	211.6	2.4	4.8	3.2	1.6
199	305	IALAAPILLAAP	12	6	57.3	204.2	2.2	4.8	3.2	1.6
200	101	LVALAPVAAVLP	12	6	57.3	203.3	2.3	4.5	3.0	1.5
201	304	AIILAPIAAIAP	12	6	57.3	204.2	2.3	4.4	3.0	1.5
202	604	VALIAVAPAVVP	12	8	57.3	195.0	2.4	4.3	2.5	1.5
203	645	ALAVVALPAIVP	12	8	50.2	203.3	2.4	4.3	2.9	1.5
204	201	LALAVPALAALP	12	6	57.3	195.8	2.1	4.2	2.8	1.4
205	163	LALVLPAALAAP	12	6	57.3	195.8	2.1	4.1	2.4	1.4
206	832	AVAAIVPVIVAP	12	7	43.2	195.0	2.5	4.1	2.7	1.4
207	182	ALIAPVVALVAP	12	6	57.3	203.3	2.4	4.0	2.7	1.4
208	23	VVLVLPAAAAVP	12	6	57.3	195.0	2.4	4.0	2.6	1.3
209	105	LLALAPAALLAP	12	6	57.3	204.1	2.1	4.0	2.6	1.3
210	561	AAVAIVLPAVVP	12	8	50.2	195.0	2.4	3.9	2.6	1.3
211	765	AVALAVVPAVLP	12	8	57.3	195.0	2.3	3.8	2.2	1.3
212	684	AAIVLALPAVLP	12	8	50.2	211.7	2.4	3.5	2.1	1.2
213	143	AVLAVPAVLVAP	12	6	57.3	195.0	2.4	3.3	2.2	1.1
214	504	LIVALAVPALAP	12	8	50.2	211.7	2.4	3.3	2.2	1.1
215	2.2	AVVLVPVLAAAP	12	6	57.3	195.0	2.4	3.1	2.1	1.1
216	5	AAALLPVALVAP	12	6	57.3	187.5	2.1	3.1	2.1	1.0
217	283	AALLAPALIVAP	12	6	50.2	195.8	2.2	3.1	2.0	1.0
218	65	IAIVAPVVALAP	12	6	50.2	203.3	2.4	3.0	2.0	1.0
219	883	LAIVPAAIAALP	12	6	50.2	195.8	2.2	3.0	2.0	1.0
220	123	AAIIVPAALLAP	12	6	50.2	195.8	2.2	2.9	2.0	1.0

[Table 30]

Sequence ID Number	aMTD	Sequences	Length	Proline Position	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)
				(PP)	(II)	(AI)	(GRAVY)	A	B	C
221	284	ALIAPAVALIVP	12	5	50.2	211.7	2.4	28	1.8	0.9
222	205	ALALVPAIAALP	12	6	57.3	195.8	2.2	2.6	1.7	0.9
223	42	VAALPWAWAP	12	5	57.3	186.7	2.4	25	1.7	0.8
224	121	AIVALP ALALAP	12	6	50.2	195.8	2.2	2.5	1.7	0.8
225	25	IVAVAPALVALP	12	6	50.2	203.3	2.4	2.4	1.6	0.8
226	24	IALAAPALIVAP	12	6	50.2	195.8	2.2	2.3	1.6	0.8
227	204	LIAALPAVAALP	12	6	57.3	195.8	2.2	2.2	1.5	0.8
228	12	LLAAVPAVLLAP	12	6	57.3	211.7	2.3	2.2	1.5	0.7
229	43	LLAAPLWAAVP	12	5	41.3	187.5	2.1	2.1	1.4	0.7
230	103	ALIAAPILALAP	12	6	57.3	204.2	2.2	2.1	1.4	0.7
231	82	AWLAPVAAVLP	12	6	57.3	195.0	2.4	2.1	1.4	0.7
232	4	ALALLPVAALAP	12	6	57.3	195.8	2.1	2.0	1.3	0.7
233	85	LLVLPAAALAAP	12	5	57.3	195.8	2.1	1.9	1.3	0.7
234	63	AALLVPALVAVP	12	6	57.3	203.3	2.3	1.9	1.3	0.7
235	44	ALAVPVALLVAP	12	5	57.3	203.3	2.3	1.6	1.1	0.5
236	84	AAVAAPLLLALP	12	6	41.3	195.8	2.1	1.5	1.0	0.5
237	62	VALLAPVALAVP	12	6	57.3	203.3	2.3	1.4	0.9	0.5
238	83	LAVAAPLALALP	12	6	41.3	195.8	2.1	1.4	0.9	0.5
239	102	LALAPAALALLP	12	5	57.3	204.2	2.1	1.4	0.9	0.5
240	623	VAAAIALPAIVP	12	8	50.2	187.5	2.3	0.8	0.6	0.3
								19.6±1.6	13.1±1.1	6.6±0.5

[0208] Moreover, compared to reference CPPs (B type: MTM12 and C type: MTD85), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively (Tables 26 to 31).

[Table 31]

	Negative Control rP38	MTM12	MTD85
aMTD The Average of 240 aMTDs	19.6 ± 1.6* (Best: 164.2)	13.1 ± 1.1* (Best: 109.9)	6.6 ± 0.5* (Best: 55.5)

"Relative Fold (aMTD in Geo Mean In its comparison to rP38, MTM12 or MTD85)

[0209] In addition, cell-permeabilities of 31 rPeptides have been compared with that of 240 aMTDs (0.3±0.04; Tables 32 and 33).

[Table 32]

Number	ID	Sequence	Length	Proline Position (PP)	Rigidity/Flexibility (II)	Sturctural Feature (AI)	Hydropathy (GRAVY)	Relative Ratio to aMTD AVE
1	692	PAPLPPVVILAV	12	1,3,5,6	105.5	186.7	1.8	0.74
2	26	AAIALAAPLAIV	12	8	18.1	204.2	2.5	0.65
3	113	PVAVALUAVPP	12	1,11,12	57.3	195.0	2.1	0.61
4	466	IIAAAAPLAIIP	12	7,12	22.8	204.2	2.3	0.52
5	167	VAIAIP AALAIP	12	6,12	20.4	195.8	2.3	0.50
6	97	ALLAAPPALLAL	12	6,7	57.3	204.2	2.1	0.41
7	390	VPLLVPVVPVVP	12	2,6,9,12	105.4	210.0	2.2	0.41
8	426	AAALAIPLAIIP	12	7,12	4.37	204.2	2.2	0.40
9	214	ALIVAPALMALP	12	6,12	60.5	187.5	2.2	0.33
10	68	VAPVLPAAPLVP	12	3,6,9,12	105.5	162.5	1.6	0.32
11	39	CYNTSPCTGCCY	12	6	52.5	0.0	0.0	0.29
12	934	LILAPAAVVAAA	12	5	57.3	195.8	2.5	0.28
13	938	VPVLLPVVVPVP	12	2,6,10,12	121.5	210.0	2.2	0.28
14	329	LPVLVPVVPVVP	12	2,6,9,12	121.5	210.0	2.2	0.23
15	606	AAAIAAIPIIIP	12	8,12	4.4	204.2	2.4	0.20
16	49	VVPAAPAVPVVP	12	3,6,9,12	121.5	145.8	1.7	0.18
17	139	TGSTNSPTCTST	12	7	53.4	0.0	-0.7	0.17
18	772	LPVAPVIPIIVP	12	2,5,8,12	79.9	210.8	2.1	0.16
19	921	IWWFVVLPLVVP	12	8,12	41.3	194.2	2.2	0.14
20	66	AGVLGGPIMGVP	12	7,12	35.5	121.7	1.3	0.13
21	693	AAPVLPVAVPIV	12	3,6,10	82.3	186.7	2.1	0.13
2.2	18	NYCCTPTTNGQS	12	6	47.9	0.0	-0.9	0.10
23	16	NNSCTTYTNGSQ	12	None	47.4	0.0	-1.4	0.08
24	227	LAAIVPIAAAVP	12	6,12	34.2	187.5	2.2	0.08
25	17	GGCSAPQTTCSN	12	6	51.6	8.3	-0.5	0.08
26	67	LDAEVPLADDVP	12	6,12	34.2	130.0	0.3	0.08
27	635	GSTGGSQQNNQY	12	None	31.9	0.0	-1.9	0.07
28	29	VLPPLPVLPVLP	12	3,4,6,9,12	121.5	202.5	1.7	0.07
29	57	QNNCNTSSQGGG	12	None	52.4	0.0	-1.6	0.06
30	700	GTSNTCQSNQNS	12	None	19.1	0.0	-1.6	0.05
31	38	YYNQSTCGGQCY	12	ND	53.8	0.0	-1.0	0.05
							AVE	0.3±0.04

[Table 33]

	Re ative Ratio to aMTD AVE*
rPeptide The Average of 31 aMTDs	0.3 ± 0.04

*Out of 240 aMTDs, average relative fold of aMTD had been 19.6 fold compared to type A (rP38).

[0210] In summary, relatively cell-permeability of aMTDs has shown maximum of 164.0, 109.9 and 55.5 fold higher to rP38, MTM12 and MTD85, respectively. In average of total 240 aMTD sequences, 19.6±1.6, 13.1±1.1 and 6.6±0.5 fold higher cell-permeability are shown to the rP38, MTM12 and MTD85, respectively (Tables 26 to 31). Relative cell-permeability of negative control (rP38) to the 240 aMTDs is only 0.3±0.04 fold.

4-5. Intracellular Delivery and Localization of aMTD-Fused Recombinant Proteins

[0211] Recombinant proteins fused to the aMTDs were tested to determine their intracellular delivery and localization by laser scanning confocal microscopy with a negative control (rP38) and previous published CPPs (MTM12 and MTD85) as the positive control references. NIH3T3 cells were exposed to 10 uM of FITC-labeled protein for 1 hour at 37°C, and nuclei were counterstained with DAPI. Then, cells were examined by confocal laser scanning microscopy (FIG. 7). Recombinant proteins fused to aMTDs clearly display intracellular delivery and cytoplasmic localization (FIG. 7) that are typically higher than the reference CPPs (MTM12 and MTD85). The rP38-fused recombinant protein did not show internalized fluorescence signal (FIG. 7a). In addition, as seen in FIG. 8, rPeptides (his-tagged CRA recombinant proteins fused to each rPeptide) display lower- or non- cell-permeability.

4-6. Summary of Quantitative and Visual Cell-Permeability of Newly Developed aMTDs

[0212] Histidine-tagged aMTD-fused cargo recombinant proteins have been greatly enhanced in their solubility and yield. Thus, FITC-conjugated recombinant proteins have also been tested to quantitate and visualize intracellular localization of the proteins and demonstrated higher cell-permeability compared to the reference CPPs.

[0213] In the previous studies using the hydrophobic signal-sequence-derived CPPs - MTS/MTM or MTDs, 17 published sequences have been identified and analyzed in various characteristics such as length, molecular weight, pI value, bending potential, rigidity, flexibility, structural feature, hydropathy, amino acid residue and composition, and secondary structure of the peptides. Based on these analytical data of the sequences, novel artificial and non-natural peptide sequences designated as advanced MTDs (aMTDs) have been invented and determined their functional activity in intracellular delivery potential with aMTD-fused recombinant proteins.

[0214] aMTD-fused recombinant proteins have promoted the ability of protein transduction into the cells compared to the recombinant proteins containing rPeptides and/or reference hydrophobic CPPs (MTM12 and MTD85). According to the results, it has been demonstrated that critical factors of cell-penetrating peptide sequences play a major role to determine peptide-mediated intracellular delivery by penetrating plasma membrane. In addition, cell-permeability can considerably be improved by following the rational that all satisfy the critical factors.

5. Structure/Sequence Activity Relationship (SAR) of aMTDs on Delivery Potential

[0215] After determining the cell-permeability of novel aMTDs, structure/sequence activity relationship (SAR) has been analyzed for each critical factor in selected some of and all of novel aMTDs (FIGs. 13 to 16 and Table 34).

[Table 34]

Rank of Delivery Potential	Rigidity/Flexibility	Sturctural Feature	Hydropathy	Relative Ratio (Fold)			Amino Acid Composition
	(II)	(AI)	(GRAVY)	A	B	C	A	V	I	L
1∼10	55.9	199.2	2.3	112.7	75.5	38.1	4.0	3.5	0.4	2.1
11∼20	51.2	205.8	2.4	56.2	37.6	19.0	4.0	2.7	1.7	1.6
21∼30	49.1	199.2	2.3	43.6	28.9	14.6	4.3	2.7	1.4	1.6
31∼40	52.7	201.0	2.4	34.8	23.3	11.8	4.2	2.7	1.5	1.6
41∼50	53.8	201.9	2.3	30.0	20.0	10.1	4.3	2.3	1.1	2.3
51∼60	51.5	205.2	2.4	23.5	15.7	7.9	4.4	2.1	1.5	2.0
222∼231	52.2	197.2	2.3	2.2	1.5	0.8	4.5	2.1	1.0	2.4
232∼241	54.1	199.7	2.2	1.7	1.2	0.6	4.6	1.7	0.2	3.5

[0216] 5-1. Proline Position: In regards to the bending potential (proline position: PP), aMTDs with its proline at 7' or 8' amino acid in their sequences have much higher cell-permeability compared to the sequences in which their proline position is at 5' or 6' (FIGs. 14a and 15a).

[0217] 5-2. Hydropathy: In addition, when the aMTDs have GRAVY (Grand Average of Hydropathy) ranging in 2.1 to 2.2, these sequences display relatively lower cell-permeability, while the aMTDs with 2.3 to 2.6 GRAVY are shown significantly higher one (FIGs. 14b and 15b).

[0218] 5-3. rPeptide SAR: To the SAR of aMTDs, rPeptides have shown similar SAR correlations in the cell-permeability, pertaining to their proline position (PP) and hydropathy (GRAVY). These results confirm that rPeptides with high GRAVY (2.4 to 2.6) have better cell-permeability (FIG. 16).

[0219] 5-4. Analysis of Amino Acid Composition: In addition to proline position and hydropathy, the difference of amino acid composition is also analyzed. Since aMTDs are designed based on critical factors, each aMTD-fused recombinant protein has equally two proline sequences in the composition. Other hydrophobic and aliphatic amino acids - alanine, isoleucine, leucine and valine - are combined to form the rest of aMTD peptide sequences.

[0220] Alanine: In the composition of amino acids, the result does not show a significant difference by the number of alanine in terms of the aMTD's delivery potential because all of the aMTDs have three to five alanines. However, in the sequences, four alanine compositions show the most effective delivery potential (geometric mean) (FIG. 13a).

[0221] Leucine and Isoleucine: Also, the compositions of isoleucine and leucine in the aMTD sequences show inverse relationship between the number of amino acid (I and L) and delivery potential of aMTDs. Lower number of isoleucine and leucine in the sequences tends to have higher delivery potential (geometric mean) (FIGs. 13a and 13b).

[0222] Valine: Conversely, the composition of valine of aMTD sequences shows positive correlation with their cell-permeability. When the number of valine in the sequence is low, the delivery potential of aMTD is also relatively low (FIG. 13b).

[0223] Ten aMTDs having the highest cell-permeability are selected (average geometric mean: 2584±126). Their average number of valine in the sequences is 3.5; 10 aMTDs having relatively low cell-permeability (average geometric mean: 80±4) had average of 1.9 valine amino acids. The average number of valine in the sequences is lowered as their cell-permeability is also lowered as shown in FIG. 13b. Compared to higher cell-permeable aMTDs group, lower sequences had average of 1.9 in their valine composition. Therefore, to obtain high cell-permeable sequence, an average of 2-4 valines should be composed in the sequence.

[0224] 5-5. Conclusion of SAR Analysis: As seen in FIG. 15, all 240 aMTDs have been examined for these associations of the cell-permeability and the critical factors: bending potential (PP), rigidity/flexibility (II), structure feature (AI), and hydropathy (GRAVY), amino acid length and composition. Through this analysis, cell-permeability of aMTDs tends to be lower when their central proline position is at 5' or 6' and GRAVY is 2.1 or lower (FIG 15). Moreover, after investigating 10 higher and 10 lower cell-permeable aMTDs, these trends are clearly shown to confirm the association of cell-permeability with the central proline position and hydropathy.

6. Experimental Confirmation of Index Range/Feature of Critical Factors

[0225] The range and feature of five out of six critical factors have been empirically and experimentally determined that are also included in the index range and feature of the critical factors initially proposed before conducting the experiments and SAR analysis. In terms of index range and feature of critical factors of newly developed 240 aMTDs, the bending potential (proline position: PP), rigidity/flexibility (Instability Index: II), structural feature (Aliphatic Index: AI), hydropathy (GRAVY), amino acid length and composition are all within the characteristics of the critical factors derived from analysis of reference hydrophobic CPPs.

[0226] Therefore, our hypothesis to design and develop new hydrophobic CPP sequences as advanced MTDs is empirically and experimentally proved and demonstrated that critical factor-based new aMTD rational design is correct.

[Table 35]

Summarized Critical Factors of aMTD
Critical Factor	Newly Designed CPPs	Analysis of Experimental Results
	Range	Range
Bending Potential (Proline Position: PP)	Proline presences in the middle (5', 6', 7' or 8') and at the end of peptides	Proline presences in the middle (5', 6', 7' or 8') and at the end of peptides
Rigidity / Flexibility (Instability Index: II)	40 - 60	41.3 - 57.3
Structural Feature (Aliphatic Index: AI)	180 - 220	187.5 - 220.0
Hydropathy (Grand Average of Hydropathy GRAVY)	2.1 - 2.6	2.2 - 2.6
Length (Number of Amino Acid)	9 - 13	12
Amino acid Composition	A, V, I, L, P	A, V, I, L, P

7. Discovery and Development of Protein-Based New Biotherapeutics with MITT Enabled by aMTDs for Protein Therapy

[0227] 240 aMTD sequences have been designed and developed based on the critical factors. Quantitative and visual cell-permeability of 240 aMTDs (hydrophobic, flexible, bending, aliphatic and 12 a/a-length peptides) are all practically determined.

[0228] To measure the cell-permeability of aMTDs, rPeptides have also been designed and tested. As seen in FIGs. 13 to 15, there are vivid association of cell-permeability and the critical factors of the peptides. Out of these critical factors, we are able to configure that the most effective cell-permeable aMTDs have the amino acid length of 12; composition of A, V, L, I and P; multiple proline located at either 7' or 8' and at the end (12'); instability index ranged of 41.3 to 57.3; aliphatic index ranged of 187.5 to 220.0; and hydropathy (GRAVY) ranged of 2.2 to 2.6.

[0229] These examined critical factors are within the range that we have set for our critical factors; therefore, we are able to confirm that the aMTDs that satisfy these critical factors have relatively high cell-permeability and much higher intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.

[0230] It has been widely evident that many human diseases are caused by proteins with deficiency or over-expression that causes mutations such as gain-of-function or loss-of-function. If biologically active proteins could be delivered for replacing abnormal proteins within a short time frame, possibly within an hour or two, in a quantitative manner, the dosage may be regulated depending on when and how proteins may be needed. By significantly improving the solubility and yield of novel aMTD according to one embodiment of the present invention (Table 31), one could expect its practical potential as an agent to effectively deliver therapeutic macromolecules such as proteins, peptides, nucleic acids, and other chemical compounds into live cells as well as live mammals including human. Therefore, newly developed MITT utilizing the pool (240) of novel aMTDs can be used as a platform technology for discovery and development of protein-based biotherapeutics to apprehend intracellular protein therapy after determining the optimal cargo-aMTD relationship.

8. Novel Hydrophobic CPPs - aMTDs for Development of iCP-Cre Recombinant Proteins 8-1. Selection of aMTD for Cell-Permeability

[0231] From 240 aMTDs, 12 aMTDs were selected and used for the construction of iCP-Cre recombinant proteins. 12 aMTDs used are shown in the following Table 36.

[0232] Various hydrophobic CPPs - aMTDs have been used to enhance the delivery of cargo (Cre) proteins to mammalian cells and tissues.

[Table 36]

SEQ ID NO	aMTD ID	Amino Acid Sequences
2	2	AAAVPLLAVVVP
17	61	VAALPVLLAALP
43	165	ALAVPVALAIVP
63	264	LAAAPVVIVIAP
131	563	ALAVIVVPALAP
134	582	VAVALIVPALAP
136	585	ALIVAIAPALVP
143	623	VAAAIALPAIVP
147	661	AAILAPIVAALP
200	847	LVAIVVLPAVAP
222	888	ILAVVAIPAAAP
229	899	AVVIALPAVVAP

8-2. Selection of Solubilization Domain (SD) for Structural Stability

[0233] Recombinant cargo (Cre) proteins fused to hydrophobic CPP could be expressed in bacteria system, purified with single-step affinity chromatography, but protein dissolved in physiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low yield as a soluble form. Therefore, an additional non-functional protein domain (solubilization domain: SD) has been applied to fuse with the recombinant protein for improving the solubility, yield and eventually cell and tissue permeability.

[0234] According to the specific aim, the selected domains are SDA to SDF (Table 37). The aMTD/SD-fused Cre recombinant proteins have been determined for their stability.

[0235] The solubilization domains (SDs) and aMTDs have greatly influenced in increasing solubility/yield and cell-/tissue-permeability of the protein. Therefore, we have developed highly soluble and highly stable Cre recombinant protein fused with SD (SDA and/or SDB) and aMTDs.

[0236] Table 37 shows the Characteristics of Solubilization Domains.

[Table 37]

SD	Genbank ID	Origin	Protein (kDa)	pI	Instability Index (II)	GRAVY
A	CP000113.1	Bacteria	23	4.6	48.1	-0.1
B	BC086945.1	Rat	11	4.9	43.2	-0.9
C	CP012127.1	Human	12	5.8	30.7	-0.1
D	CP012127.1	Bacteria	23	5.9	26.3	-0.1
E	CP011550.1	Human	11	5.3	44.4	-0.9
F	NG_034970	Human	34	7.1	56.1	-0.2

8-3. Construction of Expression Vector

[0237] 5 different types of recombinant proteins with or without the aMTD and solubilization domains (SDs) for Cre protein were designed. Protein structures were labeled as follows: (1) a Cre protein fused with His-tag, NLS, aMTD and SDB, (2) a Cre protein fused with His-tag, NLS, aMTD and SDA, (3) a Cre protein fused with His-tag, NLS, aMTD, SDA and SDB, (3-1) a Cre protein fused with His-tag and NLS only, and (3-2) a Cre protein fused with His-tag, NLS, SDA and SDB (FIGs. 17 and 20). Among them, (1) to (3) were used as candidate proteins having the biological efficacy of iCP-Cre recombinant protein, while (3-1) and (3-2) were used as control groups (Non-CP-Cre) with respect to (1) to (3).

8-4. Preparation of Cre Recombinant Proteins

[0238] The Cre recombinant proteins were successfully induced by adding IPTG and purified. The solubility and yield of the Cre recombinant proteins were determined.

[0239] Solubility will be scored on a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (*****) to largely insoluble proteins (*) by measuring their turbidity (A450). Yield (mg/L) in physiological buffer condition of each recombinant protein will also be determined.

[0240] We observed a significant increase of solubility of Cre fused with SDB (HNM₁₆₅CB) on C-terminus, which were compared to a Cre protein only (HNC) or Cre protein fused with SDA on N-terminus (HNB₅₆₃SC). And, we observed that yield and solubility of Cre protein fused with SDA and SDB on N-/C-terminus (HNM₅₆₃ACB) were greatly improved (FIGs. 19 and 22, bottom). The results suggested that the Cre recombinant proteins fused with both SDA and SDB (FIGs. 18 and 20) displayed a significant improvement of solubility and yields.

[0241] The solubility/yield, permeability, and biological activity in vitro of the Cre recombinant proteins fused with various aMTDs, as shown in FIG. 25, were measured (FIGs. 27 to 29).

[0242] By considering the solubility/yield, permeability, and biological activity measured candidate substances having the biological efficacy of the iCP-Cre recombinant protein were selected.

9. Determination of Biological Activity of Cre Recombinant Proteins with substrates

[0243] The biological activity of Cre recombinant proteins was investigated. By using two systems of assay, in the two systems, a linear or circular DNA substrate was used (FIGs. 23 and 24, top).

9-1. a Linear Substrate

[0244] The First system used a linear substrate containing an ampicillin resistant gene (FIG. 23, top). The gene in the substrate is floxed by LoxP sites. In a presence of Cre, the linear structure formed a circular form and then the gene is expressed. The ampicillin resistant gene was expressed by the Cre recombinant protein, and thus colonies were formed on a medium containing ampicillin. As a result, it was confirmed that the Cre recombinant protein recognized the LoxP sites of the substrate to show a recombination activity, a biological activity of Cre recombinant protein.

9-2. a Circular Substrate

[0245] The second system used a circular substrate containing an ampicillin resistant gene (FIG. 24, top). Since the stop sequence of the gene is located in the upstream of the gene, the gene is not expressed in an absence of Cre. However, the gene was expressed in a presence of Cre, because the stop sequence is deleted by Cre-mediated recombination in LoxP site that floxes the stop sequence. The ampicillin resistant gene was expressed by the Cre recombinant protein, and colonies were formed on a medium containing ampicillin. As a result, it was confirmed that the Cre recombinant protein recognized the LoxP sites of the substrate to show the recombination activity which is the biological activity of Cre recombinant protein.

10. Determination of Cell-, Tissue-Permeability of Cre Recombinant Proteins

[0246] The cell-/tissue-permeability of developed Cre recombinant proteins were investigated. Collectively, the aMTD/SD-fused Cre recombinant proteins (HNMAB) had significantly higher cell-, tissue-permeability as compared to the Cre recombinant proteins lacking aMTD (HNACB) or both aMTD and SD (HNC).

10-1. Cell-Permeability of Cre Recombinant Proteins

[0247] The cell-permeability of developed Cre recombinant proteins was investigated. Cre recombinant proteins was labeled fluorescence dye, FITC (fluorescein isothiocyanate), then cell permeability of the Cre recombinant proteins was evaluated in RAW 264.7 cells or NIH3T3 cells.

[0248] The RAW 264.7 cells analyzed by FACS (fluorescence-activated cell sorting) showed a gain in fluorescence, indicative of the presence of FITC-labeled proteins as compared with control that only FITC or diluent. The cells (1X10⁴) were analyzed by using the CellQues Pro cytometric analysis software (FACS Calibur, Beckton-Dickinson, San Diego CA, USA). Cell permeability of each of the Cre recombinant proteins fused with 9 aMTDs was examined (FIGs. 28a, 28b and 30).

[0249] The presence of the iCP-Cre recombinant proteins in the NIH3T3 cells was verified by confocal laser microscopy by immunocytochemistry (FIG. 31).

10-2. Tissue-Permeability of Cre Recombinant Proteins

[0250] The tissue-permeability of developed Cre recombinant proteins was investigated. Tissue-permeability of proteins was investigated by intravenous (I.V.) injection of a FITC-labeled aMTD/SD-fused Cre recombinant protein into mice. Tissues obtained from various organs (brain, heart, lung, liver, spleen, kidney, eyes and so on) after the injection of the protein show that the aMTD-/SD-fused Cre recombinant protein is delivered into each organ (FIG. 32). Thus, these results suggest that the Cre recombinant protein attaching aMTD is enhanced its tissue-permeability and therefore, aMTD is critical for systemic delivery of the protein in vivo.

11. Determination of Cell-to-Cell Delivery of Cre Recombinant Proteins

[0251] Cell-to-cell delivery of the Cre recombinant proteins, which is required for genetic recombination by the Cre recombinant proteins in vivo, was investigated.

[0252] FITC-labeled Cre protein-treated cells and Cy5.5-labeled CD14 Ab-treated cells were co-cultured, and the population of the FITC/Cy5.5-labeled cells was counted in the Cy5.5-labeled CD14 Ab-treated cells (FIG. 33, top). FACS analysis shows that cell-permeated Cre recombinant proteins were delivered to another cell (FIG. 33, bottom).

12. Determination of Biological Activity of Cre Recombinant Proteins in a reporter cell

[0253] The biological activity of Cre recombinant proteins in color-switch reporter cell line, Tex Loxp.EG was investigated. The Tex.loxp.EG is a T-lymphocyte line in which Cre-mediated recombination activates the expression of a green fluorescent protein (GFP) reporter gene (FIG. 35, top). Since the stop sequence of the EGFP gene is located in the upstream of a gene, the gene is not expressed in an absence of Cre. However, the gene was expressed in a presence of Cre, because the stop sequence is deleted by Cre-mediated recombination in LoxP site that floxes the stop sequence. FACS analysis shows that the target gene was expressed by recombination mediated by the Cre recombinant proteins (FIG. 35, bottom).

13. Determination of Biological Activity of Cre Recombinant Proteins in vivo

[0254] The biological activity of Cre recombinant proteins was investigated by using transgenic mice.

[0255] On ROSA26-LSL-lacZ and ROSA26-eYFP mice, since the stop sequence is located in the upstream of the lacZ or eYFP gene, the gene is not expressed in an absence of Cre. However, the gene was expressed in a presence of Cre, because the stop sequence is deleted by Cre-mediated recombination in LoxP site that floxes the stop sequence (FIGs. 36 and 37, top).

[0256] On SOCS3^f/f mice, since a LoxP site is located in the middle of exon 2 of SOCS3 gene, the SOCS3 gene is expressed in an absence of Cre. However, the SOCS3 gene was not expressed in a presence of Cre, because part of the SOCS3 gene is deleted (FIGs. 38, 39 and 42, top).

[0257] On ROSA^nT-nG mice, since the RFP gene is located in the upstream of the eGFP gene, the eGFP gene is not expressed in an absence of Cre. However, the eGFP gene was expressed in a presence of Cre, because the RFP gene is deleted by Cre-mediated recombination in LoxP site that floxes the RFP sequence (FIG. 43, top).

[0258] As a result, it was confirmed that the Cre recombinant proteins mediate conditional knockout of the target gene to inhibit expression of the gene.

14. Summary

[0259] According to one embodiment of the present invention, improved cell-permeable Cre recombinant proteins have been designed and developed with the aMTD and SDs. All Cre recombinant proteins fused with aMTD/SD and control recombinant proteins lacking aMTD or both aMTD and SD have been confirmed for their quantitative, visual cell-/tissue-permeability and biological activity in vitro and in vivo. Consequently, the Cre recombinant proteins fused with SD were confirmed to have relatively high solubility, cell permeability, and biological activity, and the optimized structure of the Cre recombinant proteins was determined. The optimal aMTD was also determined for the high yield, solubility, and cell-permeability of the Cre recombinant proteins. The Cre proteins fused with the optimal aMTD/SDs are iCP-Cre recombinant proteins with superior cell-/tissue-permeability and cell-to-cell delivery, compared to Cre recombinant protein lacking aMTD/SDs. It was confirmed that these iCP-Cre recombinant proteins have the Cre protein of biological activity that mediated knockout or recombination of a target gene in cells or tissues by the Cre/LoxP system.

[0260] The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols.

Example 1. Development of Novel Advanced Macromolecule Transduction Domain (aMTD)

[0261] H-regions of signal sequences (HOURSP)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, a sequence motif, and/or a common structural homologous feature. According to one embodiment of the present invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined 'critical factors' to have a 'common function,' to facilitate protein translocation across the plasma membrane with similar mechanism to the analyzed CPPs.

[0262] The structural motif as follows:

[0263] In Table 9, universal common sequence/structural motif is provided as follows. The amino acid length of the peptides according to one embodiment of the present invention ranges from 9 to 13 amino acids, mostly 12 amino acids, and their bending potentials are dependent with the presence and location of proline in the middle of sequence (at 5', 6', 7' or 8' amino acid) and at the end of peptide (at 12') for recombinant protein bending. Instability index (II) for rigidity/flexibility of aMTDs is II<40, grand average of hydropathy (GRAVY) for hydropathy is around 2.2, and aliphatic index (AI) for structural features is around 200 (Table 9). Based on these standardized critical factors, new hydrophobic peptide sequences, namely advanced macromolecule transduction domain peptides (aMTDs), according to one embodiment of the present invention have been developed and summarized in Tables 10 to 15.

Example 2. Construction of Expression Vectors for Recombinant Proteins Fused to aMTDs

[0264] Our newly developed technology has enabled us to expand the method for making cell-permeable recombinant proteins. The expression vectors were designed for histidine-tagged CRA proteins fused with aMTDs or rPeptides. To construct expression vectors for recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify each designed aMTD or rPeptide fused to CRA.

[0265] The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1X reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea) was digested on the restriction enzyme site between Nde I (5') and Sal I (3') involving 35 cycles of denaturation (95°C), annealing (62°C), and extension (72°C) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 5 minutes at 72°C. Then, they were cloned into the site of pET-28a(+) vectors (Novagen, Madison, WI, USA). DNA ligation was performed using T4 DNA ligase at 4°C overnight. These plasmids were mixed with competent cells of E. coli DH5-alpha strain on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42°C for 90 seconds. Then, the mixture added with LB broth media was recovered in 37°C shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 ug/mL) (Biopure, Johnson City, TN, USA) before incubating at 37°C overnight. From a single colony, plasmid DNA was extracted, and after the digestion of Nde I and Sal I restriction enzymes, digested DNA was confirmed at 645 bp by using 1.2% agarose gels electrophoresis (FIG. 2). PCR primers for the CRA recombinant proteins fused to aMTD and random peptides (rPeptide) are summarized in Tables 23 to 30. Amino acid sequences of aMTD and rPeptide primers are shown in Tables 31 to 38.

Example 3. Inducible Expression, Purification and Preparation of Recombinant Proteins Fused to aMTDs and rPeptides

[0266] To express recombinant proteins, pET-28a(+) vectors for the expression of CRA proteins fused to a negative control [rPeptide 38 (rP38)], reference hydrophobic CPPs (MTM12 and MTD₈₅) and aMTDs were transformed in E. coli BL21 (DE3) strains. Cells were grown at 37°C in LB medium containing kanamycin (50 ug/ml) with a vigorous shaking and induced at OD₆₀₀=0.6 by adding 0.7 mM IPTG (Biopure) for 2 hours at 37°C. Induced recombinant proteins were loaded on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (InstantBlue, Expedeon, Novexin, UK) (FIG. 3).

[0267] The E. coli cultures were harvested by centrifugation at 5,000x rpm for 10 minutes, and the supernatant was discarded. The pellet was re-suspended in the lysis buffer (50 mM NaH₂PO₄, 10 mM Imidazol, 300 mM NaCl, pH 8.0). The cell lysates were sonicated on ice using a sonicator (Sonics and Materials, Inc., Newtown, CT, USA) equipped with a probe. After centrifuging the cell lysates at 5,000 x rpm for 10 minutes to pellet the cellular debris, the supernatant was incubated with lysis buffer-equilibrated Ni-NTA resin (Qiagen, Hilden, Germany) gently by open-column system (Bio-rad, Hercules, CA, USA). After washing protein-bound resin with 200 ml wash buffer (50 mM NaH₂PO₄, 20 mM Imidazol, 300 mM NaCl, pH 8.0), the bounded proteins were eluted with elution buffer (50 mM NaH₂PO₄, 250 mM Imidazol, 300 mM NaCl, pH 8.0).

[0268] Recombinant proteins purified under natural condition were analyzed on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (FIG. 4). All of the recombinant proteins were dialyzed for 8 hours and overnight against physiological buffer, a 1:1 mixture of cell culture medium (Dulbecco's Modified Eagle's Medium: DMEM, Hyclone, Logan, UT, USA) and Dulbecco's phosphate buffered saline (DPBS, Gibco, Grand Island, NY, USA). From 316 aMTDs and 141 rPeptides cloned, 240 aMTD- and 31 rPeptide-fused recombinant proteins were induced, purified, prepared and analyzed for their cell-permeability.

Example 4. Determination of Quantitative Cell-Permeability of Recombinant Proteins

[0269] For quantitative cell-permeability, the aMTD- or rPeptide-fused recombinant proteins were conjugated to fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). RAW 264.7 cells were treated with 10 uM FITC-labeled recombinant proteins for 1 hour at 37°C°C, washed three times with cold PBS, treated with 0.25% tripsin/EDTA (Sigma-Aldrich, St. Louis, MO) for 20 minutes at 37°C°C to remove cell-surface bound proteins. Cell-permeability of these recombinant proteins were analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJo cytometric analysis software (FIGs. 5 to 6). The relative cell-permeability of aMTDs were measured and compared with the negative control (rP38) and reference hydrophobic CPPs (MTM12 and MTD85) (Table 31).

Example 5. Determination of Cell-Permeability and Intracellular Localization of Recombinant Proteins

[0270] For a visual reference of cell-permeability, NIH3T3 cells were cultured for 24 hours on coverslip in 24-wells chamber slides, treated with 10 uM FITC-conjugated recombinant proteins for 1 hour at 37°C, and washed three times with cold PBS. Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, JP) for 10 minutes at room temperature, washed three times with PBS, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA, USA), and counter stained with DAPI (4',6-diamidino-2-phenylindole). The intracellular localization of the fluorescent signal was determined by confocal laser scanning microscopy (LSM700, Zeiss, Germany; FIGs. 7 and 8).

Example 6. Construction of Expression Vectors for Recombinant Proteins

<6-1> Construction of Expression Vectors for Recombinant Proteins

[0271] Our newly developed technology, aMTD-based MITT, has enabled us to improve the method for developing cell-permeable recombinant proteins. The expression vectors were designed for Cre recombinant proteins fused with aMTD/SDs (HNM₁₆₅CB, HNM₅₆₃AC and HNM₅₆₃ACB) and control proteins without aMTD (HNC and HNACB). To acquire expression vectors for Cre recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify these recombinant proteins.

[0272] The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1X reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor Protein, Korea)) was digested on the different restriction enzyme site involving 40 cycles of denaturation (95°C), annealing (58°C), and extension (72°C) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 10 minutes at 72°C.

[0273] Histidine-tagged Cre recombinant proteins are constructed by amplifying the Cre cDNA (343 amino acids) from nt 1 to 1029, using the primers (Table 38), for aMTD/SD-fused to Cre cargo. NLS/aMTD-SDA and SDB are prepared by amplifying its templates using the primers (Table 39). The PCR products of NLS/aMTD-SDA and SDB are cleaved with NdeI/EcoRI and SalI/XhoI, respectively. The amplified and cohesive-ended NLS/aMTD-SDA are ligated to the EcoRI site of the N-terminus of Cre; and the amplified and cohesive-ended SDB are ligated to the SalI site of the C-terminus of Cre, then finally ligated into 6xHis expression vector, pET-28a(+) (Novagen, Mdison, WI, USA). In addition, NLS-Cre and NLS-SDA are amplified its template using the primers (Tables 38 and 39). The PCR products of NLS-SDA and NLS-Cre are cleaved with NdeI/EcoRI and Ndel/SalI, respectively. The amplified and cohesive-ended NLS/SDA is ligated to the EcoRI site of the N-terminus of Cre in pET-28a(+) vector inserted Cre-SDB; and the amplified and cohesive-ended NLS/Cre is ligated to the SalI site of the pET-28a(+) vector. DNA ligation was performed using T4 DNA ligase (NEB, USA) at 4 °C overnight. These plasmids were mixed with competent cells of E.coli BL21(DE3) CodonPlus-RIL strain (ATCC, USA) on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat-shocked in the water bath at 42°C for 90 seconds. Then, the mixture added with LB broth media (ELPIS, Korea) was recovered in 37°C shaking incubator for 1 hour. Then, Transformant was plated on LB broth agar plate with kanamycin (50 ug/mL) (Biopure, Johnson, TN) before incubating overnight at 37°C. From a single colony, plasmid DNA was extracted; and after the double digestion of Ndel and Xhol restriction enzymes, digested DNA was confirmed by using 1.2% agarose gels electrophoresis (FIGs. 18 and 21).

[0274] As shown in FIGs. 18 and 21, it was confirmed that the Cre recombinant proteins (HNMCB, HNMAC, HNMACB, HNC and HNACB) were expressed from the respective recombinant expression vectors.

[0275] PCR primers for the His-tagged Cre recombinant proteins fused to aMTD and SD are summarized in Tables 38 and 39.

[Table 38]

Cargo	Recombinant Protein	5' Primers (5' → 3')	3' Primer (5' → 3')
Cre	HNC
	HNMCB
	HNMAC
	HNMACB

[Table 39]

Cargo	SD	Recombinant Protein	5' Primers (5' → 3')	3' Primer (5'→ 3')
Cre	SDA	HNM563AC
		HNM563ACB
	SDB	HNMCB
		HNMACB
		HNACB

<6-2> Expression and Purification of Histidine-tagged Cre Recombinant Proteins

[0276] The transformant was cultured in LB medium containing 25 ug/ml of kanamycin, and the transformant was inoculated in 5 ml of LB medium at 37°C overnight. The incubated transformant was inoculated in 500 ml of LB medium at 37°C until OD₆₀₀ reached 0.5. The medium was added with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) as a protein expression inducer, and further incubated at 16°C for 16 hours. The medium was centrifuged at 4°C and 8,000 x g for 5 minutes, and a supernatant was discarded to recover a cell pellet. The pellet was loaded on SDS-PAGE to analyze expression levels. The pellet was suspended in a lysis buffer (50 mM Tris-HCl, pH 9.0, 300 mM NaCl) and lysozyme (Sigma aldrich) was added at a concentration of 1 mg/ml, and then allowed to react at room temperature for 1 hour. This suspension was disrupted with sonication to the cells. The disrupted cells were centrifuged at 4°C and 15,000 x g for 30 minutes to obtain a soluble fraction and an insoluble fraction. After, the soluble fraction was used for protein purification. Recombinant proteins are supposed to be purified by Co²⁺ affinity chromatography as directed by the supplier (G-Biosciences, USA) in the natural condition. After purification, they will be changed to a 50 mM Tris-HCl (pH 9.0) buffer containing 150 mM NaCl and 10% Glucose.

<6-3> Determination of Solubility/Yield of Cre Recombinant Proteins

[0277] The aMTD-fused Cre recombinant proteins containing SDA and/or SDB are cloned, expressed, purified, and prepared in a soluble form under the native condition. Each recombinant protein; HNM₁₆₅CB, HNM₅₆₃AC, HNM₅₆₃ACB, HNC and HNACB was determined for their size (number of amino acids), yield (mg/L) and solubility on 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue.

[0278] As shown in FIGs. 19 and 22 (top), the purified Cre recombinant proteins were observed as a single band, where the amount of the final purified protein was up to 30 mg/L. As shown in FIGs. 19 and 22 (bottom), It was also confirmed that HNM₅₆₃ACB showed excellent yield and solubility, compared to HNM₁₆₅CB and HNM₅₆₃AC, then, HNMAB was determined as a basic structure of the iCP-Cre recombinant protein.

Example 7. Determination of Biological Activity of Cre Recombinant Proteins in vitro

[0279] To evaluate the biological activity of the aMTD/SD-fused Cre recombinant protein (HNM₅₆₃ACB), a linear or circular DNA substrate was used. As a control, commercial Cre protein (NEB, UK) was used.

<7-1> Biological Activity with Linear Substrate

[0280] A linear DNA substrate (NEB, UK) was used (FIG. 23, left). Cre recombinant proteins (iCP-Cre, 0.1 µg) or NEB Cre (0.2 µg) were incubated with 150 µg of the substrate in 30 min at 37°C in 50 µℓ of reaction buffer (33 mM NaCl, 50 mM Tris-HCl and 10 mM MgCl₂). The mixture was incubated at 70°C for 10 minutes for inactivation, and left on ice for 5 minutes. The mixture was transformed into E. coli, and then, the colonies were observed to measure the biological activity of the proteins.

[0281] As shown in FIG. 23 (bottom), the Cre recombinant protein (iCP-Cre) showed 2-fold higher colony formation than NEB Cre. As a result, the Cre recombinant protein has an excellent biological activity, compared to NEB Cre.

<7-2> Biological Activity with Circular Substrate

[0282] A circular DNA substrate was prepared (FIG. 24, top). The circular substrate containing LoxP sites is constructed in pET-28a(+) vector. Ampicillin resistance gene cDNA was amplified using the primers (Table 56) and the PCR product was cleaved with BamHI/SalI (NEB, UK). The cohesive-ended ampicillin was ligated to BamHI/SalI site of pET-28a(+) vector. LoxP/sS3SH2 that was the stop sequence of ampicillin resistance gene was amplified using the primers and the PCR product was cleaved with NdeI/BamHI (NEB, UK). The cohesive-ended PCR product was ligated to the pET-28a(+) vector inserting the ampicillin resistance gene. After propagate of the plasmid using DH5α, plasmid DNA was extracted and stored at -70°C. The cDNA sequence of ampicillin resistance gene and the cDNA sequence of sS3SH2 were represented by Table 40.

[Table 40]

Gene	5' Primers (5' → 3')	3' Primer (5' → 3')
sS3SH2
Ampicillin Resistance Gene	CAATAAGGATCCATGAGTATTCAACATTIC	GACACGGTCGACTTACCAATGCTTAATCAG

[0283] Cre recombinant proteins (iCP-Cre 0.1 ug) or NEB Cre (0.2 ug) were incubated with 150 µg of the substrate in 30 min at 37°C in 50 µℓ of reaction buffer (33 mM NaCl, 50 mM Tris-HCl and 10 mM MgCl₂). The mixture was incubated at 70°C for 10 minutes for inactivation, and left on ice for 5 minutes. The mixture was transformed into E. coli, and then, the colonies were observed to measure the biological activity of the proteins.

[0284] As shown in FIG. 24 (bottom), even though the amount of the Cre recombinant protein (iCP-Cre) used was 1/2 of the amount of NEB Cre, the Cre recombinant protein showed 4-fold higher colony formation than NEB Cre. As a result, the Cre recombinant protein (iCP-Cre) has an excellent biological activity, compared to NEB Cre. This result suggests that Cre recombinant protein fused to aMTD/SD has a high ability for biological activity and thus, aMTD plays a critical role in the improvement of functional ability of aMTD/SD-fused Cre protein in biological approaches in vitro.

Example 8. Determination of Optimal aMTD for iCP-Cre Recombinant Proteins

[0285] For determination of optimal aMTD for the iCP-Cre recombinant proteins, yield, solubility, cell permeability, and biological activity of each of the Cre recombinant proteins fused with different aMTDs were evaluated.

<8-1> Determination of Solubility/Yield of Cre Recombinant Proteins

[0286] In the same manner as in Example <6-1>, recombinant expression vectors expressing aMTD₂, aMTD₆₁, aMTD₂₆₄, aMTD₅₆₃, aMTD₅₈₂, aMTD₅₈₅, aMTD₆₂₃, aMTD₆₆₁, aMTD₈₄₇, aMTD₈₈₈, and aMTD₈₉₉-fused Cre recombinant proteins were prepared (FIGs. 25 and 26), and primers used are as given in Table 41.

[0287] In the same manner as in Example <6-2>, each of Cre recombinant proteins was expressed and purified from the recombinant expression vectors. In the same manner as in Example <6-3>, yield and solubility of the Cre recombinant proteins were measured.

[0288] As shown in FIG. 27, all the Cre recombinant proteins fused with different aMTDs showed high solubility. The aMTD₅₆₃-fused Cre recombinant protein was found to have the highest yield and solubility.

<8-2> Determination of Cell-Permeability of Cre Recombinant Proteins

[0289] For quantitative cell permeability, the Cre recombinant proteins were conjugated to FITC according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). RAW 264.7 cells were treated with 10 uM FITC-labeled proteins for 1 hour at 37°C, and washed three times with cold PBS. The cells treated with proteinase K (10 ug/ml) for 20 min at 37°C to remove cell-surface bound proteins and subjected to fluorescence-activated cell sorting (FACS) analysis (FACSCalibur; BD, Franklin Lakes, NJ).

[0290] As shown in FIGs. 28a and 28b, aMTD-fused Cre recombinant protein (HNMACB) showed about 8-fold higher cell permeability than the Cre recombinant protein (HNC) without aMTD and SD. The aMTD₅₆₃-fused Cre recombinant protein also showed excellent cell permeability, like other aMTD-fused Cre recombinant proteins.

<8-3> Determination of Biological Activity of Cre Recombinant Proteins

[0291] To measure biological activity of the Cre recombinant proteins in vitro, the same circular DNA substrate as in Example <7-2> was used. Formation of ampicillin-resistant colonies was observed, and the number of colonies was counted to determine and compare specific activities of each of the proteins.

[0292] As shown in FIG. 29, when the aMTD₅₆₃- or aMTD₆₆₁-fused Cre recombinant protein was treated, the largest number of colonies was formed. As a result, the aMTD₅₆₃- or aMTD₆₆₁-fused Cre recombinant protein has the most excellent biological activity.

[0293] As in the following Table 42, yield, solubility, cell permeability, and biological activity of each of the Cre recombinant proteins fused with different aMTDs were compared, and the aMTD₅₆₃-fused Cre recombinant protein was determined as iCP-Cre recombinant protein.

[Table 42]

Solubility				Permeability			In Vitro Activity
Rank	aMTD	Yield (mg/L)		Rank	aMTD		Rank	aMTD
1	563	20		1	563		1	661
2	661	12		2	889		2	563
3	264	8		3	264		3	899
				4	661		4	61
4	847	6		5	585		5	264
5	582/889	5		6	847		6	888
6	585	4		7	888		7	585
7	61	3		8	582		8	847
8	888	6		9	61		9	582

Example 9. Determination of Cell-Permeability of iCP-Cre Recombinant Proteins

<9-1> Flow Cytometry

[0294] Cell permeability of the iCP-Cre recombinant proteins was measured in the same manner as in Example <8-2>.

[0295] As shown in FIG. 30, the iCP-Cre recombinant protein (HNMACB) showed about 6∼25-fold higher cell permeability than the Cre recombinant proteins without aMTD (HNC and HNACB). This result suggests that cell permeability of the Cre recombinant protein is improved by aMTD.

<9-2> Confocal Laser Microscope

[0296] To investigate cell permeability and intranuclear delivery of the iCP-Cre recombinant proteins, immunocytochemistry assay was performed.

[0297] A cover glass was sterilized with ethanol and washed with PBS, and then placed in a 12-well plate. NIH-3T3 cells were seeded and cultured therein. The cells were treated with 10 uM of the iCP-Cre recombinant protein for 2 hours, and added with 4% formaldehyde at RT for 15 minutes for cell fixation. The cells were treated with a permeabilization solution (0.5% Triton X-100) at RT for 10 minutes. Then, the cells were treated with a blocking solution (1XPBS 189 ml + 5% BSA 10 ml + 0.5 % Tween-20 1 ml) at RT for 30 to 60 minutes. A primary antibody (anti-Cre antibody) was diluted in the blocking solution (1:400) and incubated at 4°C O/N with the cells. After, a secondary antibody (Texas Red-X goat anti-rabbit IgG) was diluted in the blocking solution (1:200) and incubated at RT for 45 minutes with the cells in the dark. The cells were fixed with a mounting medium containing DAPI (4',6-diamidino-2-phenylindole), and then observed under a confocal microscope.

[0298] As shown in FIG. 31, it was found that the iCP-Cre recombinant proteins showed cell permeability as well as intranuclear delivery. These results suggest that the iCP-Cre recombinant proteins have excellent cell permeability and induce intranuclear delivery of iCP-Cre recombinant proteins to show the biological activity (recombination).

Example 10. Determination of Tissue-Permeability of iCP-Cre Recombinant Proteins

[0299] To investigate tissue permeability of the iCP-Cre recombinant proteins, the iCP-Cre recombinant proteins in the organs of mice were measured.

[0300] FITC-labeled iCP-Cre recombinant proteins (300 ug/mouse) were administered to wild type Balb/c mice by intravenous (I.V.) injection. After 2 hours, the mice are sacrificed, and the samples of organs (liver, kidney, spleen, lung, heart, brain, eye, intestine, stomach, muscle, thymus, ovary) were embedded with an OCT compound (Sakura, Alphen an den Rijn, Netherlands), frozen and then sectioned to a thickness of 14 um. The tissue specimens were mounted on a glass slide and observed by fluorescence microscopy (Nikon, Tokyo, Japan).

[0301] As shown in FIG. 32, the iCP-Cre recombinant proteins were observed in all organs of the mice. These results suggest that the Cre recombinant protein fused aMTD is enhanced its tissue-permeability and therefore, aMTD is critical for systemic delivery of the protein in vivo.

Example 11. Determination of Cell-to-Cell Delivery of iCP-Cre Recombinant Proteins

[0302] To investigate cell-to-cell delivery of the iCP-Cre recombinant proteins, which is required for recombination in vivo, RAW 264.7 cells treated with 10 uM of FITC-labeled iCP-Cre recombinant protein and RAW 264.7 cells treated with Cy5.5 labeled-CD14 Ab were co-cultured, and changes in the population of the double-positive (Cy5.5 and FITC labeled) cells were analyzed by FACS.

[0303] As shown in FIG. 33 (bottom), the cells treated with Cy5.5-labeled CD14 Ab showed higher populations after co-culture with the cells treated with FITC-labeled iCP-Cre recombinant protein than before co-culture therewith. These results suggest that Cre recombinant proteins have cell-to-cell delivery, namely, tissue-permeability. Further, the iCP-Cre recombinant proteins are effectively delivered to each organ to mediate recombination in vivo.

Example 12. Determination of Biological Activity of iCP-Cre Recombinant Proteins in a Dose Dependent Manner

[0304] To investigate the dose-dependent biological activity of the iCP-Cre recombinant proteins, the biological activity was measured in the same manner as in Example <7-2>.

[0305] After, the mixture was incubated at 70°C for 10 minutes for inactivation, and left on ice for 5 minutes. The mixture was transformed into E. coli, and then, the colonies are observed to measure the biological activity of the proteins.

[0306] As shown in FIG. 34, when 10 to 500 ng of the iCP-Cre recombinant protein was treated, colony formation was observed. 200 ng of the iCP-Cre recombinant protein showed the most excellent biological activity.

Example 13. Determination of Biological Activity of iCP-Cre Recombinant Proteins in Reporter Cells

[0307] To investigate the biological activity of the iCP-Cre recombinant proteins at a cell level, Tex.LoxP.EG cells were used as color-switch reporter cells (containing LoxP sites) (FIG. 35, top).

[0308] The Tex.LoxP.EG is a T-lymphocyte line in which Cre-mediated recombination activates the expression of a green fluorescent protein (GFP) reporter gene. The cells were treated with 10 uM of the iCP-Cre recombinant protein for 2 hours at 37°C. After 24 hours, GFP expression levels were measured by FACS.

[0309] As shown in FIG. 35 (bottom), the cells showed 80% or more of EGFP expression by the iCP-Cre recombinant protein. As a result, it was confirmed that the iCP-Cre recombinant protein deletes the target gene in the nucleus by the Cre/LoxP system.

Example 14. Determination of Biological Activity of iCP-Cre Recombinant Proteins in vivo

[0310] To investigate the recombination activity of the iCP-Cre recombinant proteins in vivo, 4 transgenic mice were used.

<14-1> ROSA26-LSL-LacZ Mouse

[0311] The ROSA26-LSL-LacZ mice were administered with iCP-Cre recombinant protein (24 mg/kg/day) or buffer intravenously for five consecutive days. After 2 days, the mice are sacrificed, and the organs (brain, lung, liver, heat, kidney, spleen, intestine, colon and fat) were collected. The tissue samples were embedded with an OCT compound, frozen and then sectioned to a thickness of 14 uM. The tissue specimens were mounted on a glass slide. The organs/tissues were subjected to X-gal staining.

[0312] As shown in FIG. 36 (bottom), β-galactosidase expression was observed in the organs and tissues of the ROSA26-LSL-LacZ mice administered with the iCP-Cre recombinant protein.

<14-2> ROSA26-eYFP Mouse

[0313] The ROSA26-eYFP mice were treated with iCP-Cre recombinant protein (24 mg/kg/day) or buffer intravenously injection for five consecutive days and sacrificed 2 days later. The mice were sacrificed, and the organs (stomach, muscle, kidney, spleen, lung, colon, testis, liver, brain and heart) were collected. The tissue samples were embedded with an OCT compound, frozen and then sectioned to a thickness of 14 uM. The tissue specimens were mounted on a glass slide. The tissues were observed under a fluorescence microscope.

[0314] As shown in FIG. 37 (bottom), yellow fluorescence protein (YFP) expression was observed in the tissues of the ROSA26-eYFP mice treated with the iCP-Cre recombinant protein.

<14-3> SOCS3 f/f Mouse

[0315] SOCS3^f/f mice were treated with iCP-Cre recombinant protein (1, 2, 4, 6, 10 mg/kg/day) or buffer by potal vein injection for 1 day. After 2 days, the mice are sacrificed, and the organs (brain, liver, stomach, kidney, pancreas, muscle, lung, colon, eye, breast and intestine) are collected. mRNA and protein were isolated from the tissue samples, and changes in the gene expressions by recombination of the target gene were examined by RT-PCR and western blot analysis.

[0316] mRNA was isolated from the tissue samples using Hybrid-RTM kit (GeneAll, Korea), and cDNA was synthesized from 1 µg of mRNA. The PCR reactions (50 ng cDNA, 10 pmol each primer, AccuPower® RT PreMix (Bioneer, Korea) was involving 30 cycles of denaturation (94°C) for 20 seconds, annealing (60°C) for 30 seconds, and extension (72°C) for 1 minute. For the last extension cycle, the PCR reactions remained for 5 minutes at 72°C.

[0317] And, Tissue samples were lysed in PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Korea) and centrifuged at 13,000 rpm for 10 minutes at 4°C. Equal amounts of lysates were separated on 12% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked using 5% skim milk or 5% albumin in TBST and incubated with the following antibodies: anti-SOCS3 primary antibody (Cell Signaling Technology), then HRP conjugated anti-mouse or anti-rabbit secondary antibody.

[0318] As shown in FIGs. 38 and 39 (bottom), both SOCS3 mRNA and protein expressions were inhibited in the organs of the SOCS3^f/f mice treated with 12 mg/kg/day of the iCP-Cre recombinant protein.

[0319] As shown in FIGs. 40 and 41, the expressions of SOCS3 mRNA and protein were inhibited depending on the administration concentration of the iCP-Cre recombinant protein in the organs of the ROSA26-eYFP mice.

[0320] As a result, gene recombination may be effectively induced by the iCP-Cre recombinant protein even at a low concentration, suggesting that recombination by the iCP-Cre recombinant protein occurs in a high efficiency.

[0321] To investigate the tissue/organ-specific recombination, SOCS3^f/f mice were administered with iCP-Cre recombinant protein (4 mg/kg/day) or buffer by portal vein or intrarenal injection for 1 day. After 2 days, the mice are sacrificed, and the organs (brain, spleen, liver, lung and kidney) are collected. mRNA was isolated from the tissue samples, and changes in the gene expressions by recombination of the target gene were examined by RT-PCR.

[0322] As shown in FIG. 42 (bottom), SOCS3 mRNA expression was inhibited in the liver of the SOCS3^f/f mice by portal vein injection with the iCP-Cre recombinant protein, and inhibited in the kidney of the SOCS3^f/f mice by intrarenal injection with the iCP-Cre recombinant protein.

[0323] The results suggest that it is possible to induce a tissue/organ-specific recombination depending to route of administration with the iCP-Cre recombinant protein.

<14-4> ROSA nT-nG Mouse

[0324] ROSA^nT-nG mice were treated with iCP-Cre recombinant protein (12 mg/kg/day) or buffer intravenously for five consecutive days. After 2 days, the mice were sacrificed, and the organs were collected. Proteins were isolated from the tissue samples, and then changes in the expressions by recombination of the target gene were examined by western blot analysis.

[0325] As shown in FIG. 43 (bottom), GFP expression was observed in all organs of the ROSA^nT-nG mice treated with the iCP-Cre recombinant protein.

[0326] Taken together, the results suggest that it is possible to produce a conditional knock mouse in which the activity of the target gene is inhibited by the iCP-Cre recombinant protein.

<110> JO, Daewoong Cellivery Therapeutics, Inc.

<120> Improved Cell-Permeable Cre (iCP-Cre) Recombinant Protein and Use Thereof

<130> FPC160035-PC

<150> US 62/202,990
<151> 2015-08-10

<160> 835

<170> KopatentIn 2.0

<210> 1
<211> 12
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<220>
<223> Amino acid Sequence of aMTD1

<400> 1

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<223> Amino acid Sequence of aMTD2

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<223> Amino acid Sequence of aMTD3

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<223> Amino acid Sequence of aMTD4

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<223> Amino acid Sequence of aMTD5

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<223> Amino acid Sequence of aMTD11

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<223> Amino acid Sequence of aMTD12

<400> 7

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<223> Amino acid Sequence of aMTD13

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<223> Amino acid Sequence of aMTD21

<400> 9

<210> 10
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<220>
<223> Amino acid Sequence of aMTD22

<400> 10

<210> 11
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<223> Amino acid Sequence of aMTD23

<400> 11

<210> 12
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<223> Amino acid Sequence of aMTD24

<400> 12

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<223> Amino acid Sequence of aMTD25

<400> 13

<210> 14
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<220>
<223> Amino acid Sequence of aMTD42

<400> 14

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<223> Amino acid Sequence of aMTD43

<400> 15

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<223> Amino acid Sequence of aMTD44

<400> 16

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<223> Amino acid Sequence of aMTD61

<400> 17

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<223> Amino acid Sequence of aMTD62

<400> 18

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<223> Amino acid Sequence of aMTD63

<400> 19

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<220>
<223> Amino acid Sequence of aMTD64

<400> 20

<210> 21
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<220>
<223> Amino acid Sequence of aMTD65

<400> 21

<210> 22
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<220>
<223> Amino acid Sequence of aMTD81

<400> 22

<210> 23
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<220>
<223> Amino acid Sequence of aMTD82

<400> 23

<210> 24
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<220>
<223> Amino acid Sequence of aMTD83

<400> 24

<210> 25
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<220>
<223> Amino acid Sequence of aMTD84

<400> 25

<210> 26
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<220>
<223> Amino acid Sequence of aMTD85

<400> 26

<210> 27
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<220>
<223> Amino acid Sequence of aMTD101

<400> 27

<210> 28
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<220>
<223> Amino acid Sequence of aMTD102

<400> 28

<210> 29
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<220>
<223> Amino acid Sequence of aMTD103

<400> 29

<210> 30
<211> 12
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<220>
<223> Amino acid Sequence of aMTD104

<400> 30

<210> 31
<211> 12
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<220>
<223> Amino acid Sequence of aMTD105

<400> 31

<210> 32
<211> 12
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<220>
<223> Amino acid Sequence of aMTD121

<400> 32

<210> 33
<211> 12
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<220>
<223> Amino acid Sequence of aMTD123

<400> 33

<210> 34
<211> 12
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<220>
<223> Amino acid Sequence of aMTD124

<400> 34

<210> 35
<211> 12
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<220>
<223> Amino acid Sequence of aMTD141

<400> 35

<210> 36
<211> 12
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<220>
<223> Amino acid Sequence of aMTD143

<400> 36

<210> 37
<211> 12
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<220>
<223> Amino acid Sequence of aMTD144

<400> 37

<210> 38
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<220>
<223> Amino acid Sequence of aMTD145

<400> 38

<210> 39
<211> 12
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<220>
<223> Amino acid Sequence of aMTD161

<400> 39

<210> 40
<211> 12
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<220>
<223> Amino acid Sequence of aMTD162

<400> 40

<210> 41
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD163

<400> 41

<210> 42
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD164

<400> 42

<210> 43
<211> 12
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<220>
<223> Amino acid Sequence of aMTD165

<400> 43

<210> 44
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD182

<400> 44

<210> 45
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD183

<400> 45

<210> 46
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD184

<400> 46

<210> 47
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD185

<400> 47

<210> 48
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD201

<400> 48

<210> 49
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD204

<400> 49

<210> 50
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD205

<400> 50

<210> 51
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD221

<400> 51

<210> 52
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD222

<400> 52

<210> 53
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD223

<400> 53

<210> 54
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD224

<400> 54

<210> 55
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD225

<400> 55

<210> 56
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD241

<400> 56

<210> 57
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD242

<400> 57

<210> 58
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD243

<400> 58

<210> 59
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD245

<400> 59

<210> 60
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD261

<400> 60

<210> 61
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD262

<400> 61

<210> 62
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD263

<400> 62

<210> 63
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD264

<400> 63

<210> 64
<211> 12
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<220>
<223> Amino acid Sequence of aMTD265

<400> 64

<210> 65
<211> 12
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<220>
<223> Amino acid Sequence of aMTD281

<400> 65

<210> 66
<211> 12
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<220>
<223> Amino acid Sequence of aMTD282

<400> 66

<210> 67
<211> 12
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<220>
<223> Amino acid Sequence of aMTD283

<400> 67

<210> 68
<211> 12
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<220>
<223> Amino acid Sequence of aMTD284

<400> 68

<210> 69
<211> 12
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<220>
<223> Amino acid Sequence of aMTD285

<400> 69

<210> 70
<211> 12
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<220>
<223> Amino acid Sequence of aMTD301

<400> 70

<210> 71
<211> 12
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<220>
<223> Amino acid Sequence of aMTD302

<400> 71

<210> 72
<211> 12
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<220>
<223> Amino acid Sequence of aMTD304

<400> 72

<210> 73
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<220>
<223> Amino acid Sequence of aMTD305

<400> 73

<210> 74
<211> 12
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<220>
<223> Amino acid Sequence of aMTD321

<400> 74

<210> 75
<211> 12
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<220>
<223> Amino acid Sequence of aMTD322

<400> 75

<210> 76
<211> 12
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<220>
<223> Amino acid Sequence of aMTD323

<400> 76

<210> 77
<211> 12
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<220>
<223> Amino acid Sequence of aMTD324

<400> 77

<210> 78
<211> 12
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<220>
<223> Amino acid Sequence of aMTD325

<400> 78

<210> 79
<211> 12
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<220>
<223> Amino acid Sequence of aMTD341

<400> 79

<210> 80
<211> 12
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<220>
<223> Amino acid Sequence of aMTD342

<400> 80

<210> 81
<211> 12
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<220>
<223> Amino acid Sequence of aMTD343

<400> 81

<210> 82
<211> 12
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<220>
<223> Amino acid Sequence of aMTD345

<400> 82

<210> 83
<211> 12
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<220>
<223> Amino acid Sequence of aMTD361

<400> 83

<210> 84
<211> 12
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<220>
<223> Amino acid Sequence of aMTD363

<400> 84

<210> 85
<211> 12
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<220>
<223> Amino acid Sequence of aMTD364

<400> 85

<210> 86
<211> 12
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<220>
<223> Amino acid Sequence of aMTD365

<400> 86

<210> 87
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD381

<400> 87

<210> 88
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD382

<400> 88

<210> 89
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD383

<400> 89

<210> 90
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD384

<400> 90

<210> 91
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD385

<400> 91

<210> 92
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD401

<400> 92

<210> 93
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD402

<400> 93

<210> 94
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD403

<400> 94

<210> 95
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD404

<400> 95

<210> 96
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD405

<400> 96

<210> 97
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD421

<400> 97

<210> 98
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD422

<400> 98

<210> 99
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD424

<400> 99

<210> 100
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD425

<400> 100

<210> 101
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD442

<400> 101

<210> 102
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD443

<400> 102

<210> 103
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD444

<400> 103

<210> 104
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD445

<400> 104

<210> 105
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD461

<400> 105

<210> 106
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD462

<400> 106

<210> 107
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD463

<400> 107

<210> 108
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD464

<400> 108

<210> 109
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD465

<400> 109

<210> 110
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD481

<400> 110

<210> 111
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD482

<400> 111

<210> 112
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD483

<400> 112

<210> 113
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD484

<400> 113

<210> 114
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD485

<400> 114

<210> 115
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD501

<400> 115

<210> 116
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD502

<400> 116

<210> 117
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD503

<400> 117

<210> 118
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD504

<400> 118

<210> 119
<211> 12
<212> PRT
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<220>
<223> Amino acid Sequence of aMTD505

<400> 119

<210> 120
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD521

<400> 120

<210> 121
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD522

<400> 121

<210> 122
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD524

<400> 122

<210> 123
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD525

<400> 123

<210> 124
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD541

<400> 124

<210> 125
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD542

<400> 125

<210> 126
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD543

<400> 126

<210> 127
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD544

<400> 127

<210> 128
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD545

<400> 128

<210> 129
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD561

<400> 129

<210> 130
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD562

<400> 130

<210> 131
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD563

<400> 131

<210> 132
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD564

<400> 132

<210> 133
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD565

<400> 133

<210> 134
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD582

<400> 134

<210> 135
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD583

<400> 135

<210> 136
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD585

<400> 136

<210> 137
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD601

<400> 137

<210> 138
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD602

<400> 138

<210> 139
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD603

<400> 139

<210> 140
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD604

<400> 140

<210> 141
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD605

<400> 141

<210> 142
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD622

<400> 142

<210> 143
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD623

<400> 143

<210> 144
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD625

<400> 144

<210> 145
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD643

<400> 145

<210> 146
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD645

<400> 146

<210> 147
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD661

<400> 147

<210> 148
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD664

<400> 148

<210> 149
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD665

<400> 149

<210> 150
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD666

<400> 150

<210> 151
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD667

<400> 151

<210> 152
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD683

<400> 152

<210> 153
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD684

<400> 153

<210> 154
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD685

<400> 154

<210> 155
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD686

<400> 155

<210> 156
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD687

<400> 156

<210> 157
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD703

<400> 157

<210> 158
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD705

<400> 158

<210> 159
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD706

<400> 159

<210> 160
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD707

<400> 160

<210> 161
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD724

<400> 161

<210> 162
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD725

<400> 162

<210> 163
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD726

<400> 163

<210> 164
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD727

<400> 164

<210> 165
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD743

<400> 165

<210> 166
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD744

<400> 166

<210> 167
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD746

<400> 167

<210> 168
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD747

<400> 168

<210> 169
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD763

<400> 169

<210> 170
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD764

<400> 170

<210> 171
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD765

<400> 171

<210> 172
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD766

<400> 172

<210> 173
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD767

<400> 173

<210> 174
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD783

<400> 174

<210> 175
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD784

<400> 175

<210> 176
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD786

<400> 176

<210> 177
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD787

<400> 177

<210> 178
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD788

<400> 178

<210> 179
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD803

<400> 179

<210> 180
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD805

<400> 180

<210> 181
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD806

<400> 181

<210> 182
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD807

<400> 182

<210> 183
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD808

<400> 183

<210> 184
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD809

<400> 184

<210> 185
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD810

<400> 185

<210> 186
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD811

<400> 186

<210> 187
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD824

<400> 187

<210> 188
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD825

<400> 188

<210> 189
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD826

<400> 189

<210> 190
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD827

<400> 190

<210> 191
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD828

<400> 191

<210> 192
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD829

<400> 192

<210> 193
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD830

<400> 193

<210> 194
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD831

<400> 194

<210> 195
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD832

<400> 195

<210> 196
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD843

<400> 196

<210> 197
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD844

<400> 197

<210> 198
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD845

<400> 198

<210> 199
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD846

<400> 199

<210> 200
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD847

<400> 200

<210> 201
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD848

<400> 201

<210> 202
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD849

<400> 202

<210> 203
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD850

<400> 203

<210> 204
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD851

<400> 204

<210> 205
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD852

<400> 205

<210> 206
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD863

<400> 206

<210> 207
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD864

<400> 207

<210> 208
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD865

<400> 208

<210> 209
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD867

<400> 209

<210> 210
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD868

<400> 210

<210> 211
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD870

<400> 211

<210> 212
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD872

<400> 212

<210> 213
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD875

<400> 213

<210> 214
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD877

<400> 214

<210> 215
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD878

<400> 215

<210> 216
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD879

<400> 216

<210> 217
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD881

<400> 217

<210> 218
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD882

<400> 218

<210> 219
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD883

<400> 219

<210> 220
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD885

<400> 220

<210> 221
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD887

<400> 221

<210> 222
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD888

<400> 222

<210> 223
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD889

<400> 223

<210> 224
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD891

<400> 224

<210> 225
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD893

<400> 225

<210> 226
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD895

<400> 226

<210> 227
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD896

<400> 227

<210> 228
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD897

<400> 228

<210> 229
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD899

<400> 229

<210> 230
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD900

<400> 230

<210> 231
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD901

<400> 231

<210> 232
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD902

<400> 232

<210> 233
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD904

<400> 233

<210> 234
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD905

<400> 234

<210> 235
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD906

<400> 235

<210> 236
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD907

<400> 236

<210> 237
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD908

<400> 237

<210> 238
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD910

<400> 238

<210> 239
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD911

<400> 239

<210> 240
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD912

<400> 240

<210> 241
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD1

<400> 241
gcggcggcgc tggcgccggt ggtgctggcg ctgccg   36

<210> 242
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD2

<400> 242
gcggcggcgg tgccgctgct ggcggtggtg gtgccg   36

<210> 243
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD3

<400> 243
gcggcgctgc tggtgccggc ggcggtgctg gcgccg   36

<210> 244
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD4

<400> 244
gcgctggcgc tgctgccggt ggcggcgctg gcgccg   36

<210> 245
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD5

<400> 245
gcggcggcgc tgctgccggt ggcgctggtg gcgccg   36

<210> 246
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD11

<400> 246
gtggtggcgc tggcgccggc gctggcggcg ctgccg   36

<210> 247
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD12

<400> 247
ctgctggcgg cggtgccggc ggtgctgctg gcgccg   36
<210> 248
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD13

<400> 248
gcggcggcgc tggtgccggt ggtggcgctg ctgccg 36

<210> 249
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD21

<400> 249
gcggtggcgc tgctgccggc gctgctggcg gtgccg 36

<210> 250
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD22

<400> 250
gcggtggtgc tggtgccggt gctggcggcg gcgccg 36

<210> 251
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD23

<400> 251
gtggtgctgg tgctgccggc ggcggcggcg gtgccg 36

<210> 252
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD24

<400> 252
attgcgctgg cggcgccggc gctgattgtg gcgccg   36

<210> 253
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD25

<400> 253
attgtggcgg tggcgccggc gctggtggcg ctgccg   36

<210> 254
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD42

<400> 254
gtggcggcgc tgccggtggt ggcggtggtg gcgccg   36

<210> 255
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD43

<400> 255
ctgctggcgg cgccgctggt ggtggcggcg gtgccg   36

<210> 256
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD44

<400> 256
gcgctggcgg tgccggtggc gctgctggtg gcgccg   36

<210> 257
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD61

<400> 257
gtggcggcgc tgccggtgct gctggcggcg ctgccg   36

<210> 258
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD62

<400> 258
gtggcgctgc tggcgccggt ggcgctggcg gtgccg   36

<210> 259
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD63

<400> 259
gcggcgctgc tggtgccggc gctggtggcg gtgccg   36

<210> 260
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD64

<400> 260
gcgattgtgg cgctgccggt ggcggtgctg gcgccg   36

<210> 261
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD65

<400> 261
attgcgattg tggcgccggt ggtggcgctg gcgccg   36

<210> 262
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD81

<400> 262
gcggcgctgc tgccggcgct ggcggcgctg ctgccg   36

<210> 263
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD82

<400> 263
gcggtggtgc tggcgccggt ggcggcggtg ctgccg   36

<210> 264
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD83

<400> 264
ctggcggtgg cggcgccgct ggcgctggcg ctgccg   36

<210> 265
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD84

<400> 265
gcggcggtgg cggcgccgct gctgctggcg ctgccg   36

<210> 266
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD85

<400> 266
ctgctggtgc tgccggcggc ggcgctggcg gcgccg   36

<210> 267
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD101

<400> 267
ctggtggcgg tggcgccggt ggcggcggtg ctgccg   36

<210> 268
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD102

<400> 268
ctggcgctgg cgccggcggc gctggcgctg ctgccg   36

<210> 269
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD103

<400> 269
gcgctgattg cggcgccgat tctggcgctg gcgccg   36

<210> 270
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD104

<400> 270
gcggtggtgg cggcgccgct ggtgctggcg ctgccg   36

<210> 271
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD105

<400> 271
ctgctggcgc tggcgccggc ggcgctgctg gcgccg   36

<210> 272
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD121

<400> 272
gcgattgtgg cgctgccggc gctggcgctg gcgccg   36

<210> 273
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD123

<400> 273
gcggcgatta ttgtgccggc ggcgctgctg gcgccg   36

<210> 274
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD124

<400> 274
attgcggtgg cgctgccggc gctgattgcg gcgccg   36

<210> 275
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD141

<400> 275
gcggtgattg tgctgccggc gctggcggtg gcgccg   36

<210> 276
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD143

<400> 276
gcggtgctgg cggtgccggc ggtgctggtg gcgccg   36
<210> 277
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD144

<400> 277
gtgctggcga ttgtgccggc ggtggcgctg gcgccg   36

<210> 278
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD145

<400> 278
ctgctggcgg tggtgccggc ggtggcgctg gcgccg   36

<210> 279
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD161

<400> 279
gcggtgattg cgctgccggc gctgattgcg gcgccg   36

<210> 280
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD162

<400> 280
gcggtggtgg cgctgccggc ggcgctgatt gtgccg   36

<210> 281
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD163

<400> 281
ctggcgctgg tgctgccggc ggcgctggcg gcgccg   36
<210> 282
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD164

<400> 282
ctggcggcgg tgctgccggc gctgctggcg gcgccg 36

<210> 283
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD165

<400> 283
gcgctggcgg tgccggtggc gctggcgatt gtgccg   36

<210> 284
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD182

<400> 284
gcgctgattg cgccggtggt ggcgctggtg gcgccg   36

<210> 285
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD183

<400> 285
ctgctggcgg cgccggtggt gattgcgctg gcgccg   36

<210> 286
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD184

<400> 286
ctggcggcga ttgtgccggc gattattgcg gtgccg   36

<210> 287
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD185

<400> 287
gcggcgctgg tgctgccgct gattattgcg gcgccg   36

<210> 288
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD201

<400> 288
ctggcgctgg cggtgccggc gctggcggcg ctgccg   36

<210> 289
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD204

<400> 289
ctgattgcgg cgctgccggc ggtggcggcg ctgccg   36

<210> 290
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD205

<400> 290
gcgctggcgc tggtgccggc gattgcggcg ctgccg   36

<210> 291
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD221

<400> 291
gcggcgattc tggcgccgat tgtggcgctg gcgccg   36

<210> 292
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD222

<400> 292
gcgctgctga ttgcgccggc ggcggtgatt gcgccg   36

<210> 293
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD223

<400> 293
gcgattctgg cggtgccgat tgcggtggtg gcgccg   36

<210> 294
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD224

<400> 294
attctggcgg cggtgccgat tgcgctggcg gcgccg   36

<210> 295
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD225

<400> 295
gtggcggcgc tgctgccggc ggcggcggtg ctgccg   36

<210> 296
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD241

<400> 296
gcggcggcgg tggtgccggt gctgctggtg gcgccg   36

<210> 297
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD242

<400> 297
gcggcgctgc tggtgccggc gctggtggcg gcgccg   36

<210> 298
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD243

<400> 298
gcggcggtgc tgctgccggt ggcgctggcg gcgccg   36

<210> 299
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD245

<400> 299
gcggcggcgc tggcgccggt gctggcgctg gtgccg   36

<210> 300
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD261

<400> 300
ctggtgctgg tgccgctgct ggcggcggcg gcgccg   36

<210> 301
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD262

<400> 301
gcgctgattg cggtgccggc gattattgtg gcgccg   36

<210> 302
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD263

<400> 302
gcgctggcgg tgattccggc ggcggcgatt ctgccg 36

<210> 303
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD264

<400> 303
ctggcggcgg cgccggtggt gattgtgatt gcgccg   36

<210> 304
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD265

<400> 304
gtgctggcga ttgcgccgct gctggcggcg gtgccg   36

<210> 305
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD281

<400> 305
gcgctgattg tgctgccggc ggcggtggcg gtgccg   36

<210> 306
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD282

<400> 306
gtgctggcgg tggcgccggc gctgattgtg gcgccg   36

<210> 307
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD283

<400> 307
gcggcgctgc tggcgccggc gctgattgtg gcgccg   36

<210> 308
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD284

<400> 308
gcgctgattg cgccggcggt ggcgctgatt gtgccg   36

<210> 309
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD285

<400> 309
gcgattgtgc tgctgccggc ggcggtggtg gcgccg   36

<210> 310
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD301

<400> 310
gtgattgcgg cgccggtgct ggcggtgctg gcgccg   36
<210> 311
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD302

<400> 311
ctggcgctgg cgccggcgct ggcgctgctg gcgccg   36

<210> 312
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD304

<400> 312
gcgattattc tggcgccgat tgcggcgatt gcgccg   36

<210> 313
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD305

<400> 313
attgcgctgg cggcgccgat tctgctggcg gcgccg   36

<210> 314
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD321

<400> 314
attgtggcgg tggcgctgcc ggcgctggcg gtgccg   36

<210> 315
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD322

<400> 315
gtggtggcga ttgtgctgcc ggcgctggcg gcgccg   36

<210> 316
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD323

<400> 316
attgtggcgg tggcgctgcc ggtggcgctg gcgccg   36

<210> 317
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD324

<400> 317
attgtggcgg tggcgctgcc ggcggcgctg gtgccg   36

<210> 318
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD325

<400> 318
attgtggcgg tggcgctgcc ggcggtggcg ctgccg   36

<210> 319
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD341

<400> 319
attgtggcgg tggcgctgcc ggcggtgctg gcgccg   36

<210> 320
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD342

<400> 320
gtgattgtgg cgctggcgcc ggcggtgctg gcgccg   36

<210> 321
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD343

<400> 321
attgtggcgg tggcgctgcc ggcgctggtg gcgccg   36

<210> 322
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD345

<400> 322
gcgctgctga ttgtggcgcc ggtggcggtg gcgccg   36

<210> 323
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD361

<400> 323
gcggtggtga ttgtggcgcc ggcggtgatt gcgccg   36

<210> 324
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD363

<400> 324
gcggtgctgg cggtggcgcc ggcgctgatt gtgccg   36

<210> 325
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD364

<400> 325
ctggtggcgg cggtggcgcc ggcgctgatt gtgccg   36

<210> 326
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD365

<400> 326
gcggtgattg tggtggcgcc ggcgctgctg gcgccg   36

<210> 327
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD381

<400> 327
gtggtggcga ttgtgctgcc ggcggtggcg gcgccg   36

<210> 328
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD382

<400> 328
gcggcggcgc tggtgattcc ggcgattctg gcgccg   36

<210> 329
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD383

<400> 329
gtgattgtgg cgctggcgcc ggcgctgctg gcgccg   36

<210> 330
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD384

<400> 330
gtgattgtgg cgattgcgcc ggcgctgctg gcgccg   36

<210> 331
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD385

<400> 331
attgtggcga ttgcggtgcc ggcgctggtg gcgccg   36

<210> 332
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD401

<400> 332
gcggcgctgg cggtgattcc ggcggcgatt ctgccg   36

<210> 333
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD402

<400> 333
gcgctggcgg cggtgattcc ggcggcgatt ctgccg   36

<210> 334
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD403

<400> 334
gcggcggcgc tggtgattcc ggcggcgatt ctgccg   36

<210> 335
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD404

<400> 335
ctggcggcgg cggtgattcc ggcggcgatt ctgccg   36

<210> 336
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD405

<400> 336
ctggcggcgg cggtgattcc ggtggcgatt ctgccg   36

<210> 337
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD421

<400> 337
gcggcgattc tggcggcgcc gctgattgcg gtgccg   36

<210> 338
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD422

<400> 338
gtggtggcga ttctggcgcc gctgctggcg gcgccg   36

<210> 339
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD424

<400> 339
gcggtggtgg tggcggcgcc ggtgctggcg ctgccg   36
<210> 340
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD425

<400> 340
gcggtggtgg cgattgcgcc ggtgctggcg ctgccg   36

<210> 341
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD442

<400> 341
gcgctggcgg cgctggtgcc ggcggtgctg gtgccg   36

<210> 342
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD443

<400> 342
gcgctggcgg cgctggtgcc ggtggcgctg gtgccg   36

<210> 343
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD444

<400> 343
ctggcggcgg cgctggtgcc ggtggcgctg gtgccg   36

<210> 344
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD445

<400> 344
gcgctggcgg cgctggtgcc ggcgctggtg gtgccg   36
<210> 345
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD461

<400> 345
attgcggcgg tgattgtgcc ggcggtggcg ctgccg   36

<210> 346
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD462

<400> 346
attgcggcgg tgctggtgcc ggcggtggcg ctgccg   36

<210> 347
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD463

<400> 347
gcggtggcga ttctggtgcc gctgctggcg gcgccg   36

<210> 348
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD464

<400> 348
gcggtggtga ttctggtgcc gctggcggcg gcgccg   36

<210> 349
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD465

<400> 349
attgcggcgg tgattgtgcc ggtggcggcg ctgccg   36

<210> 350
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD481

<400> 350
gcgattgcga ttgcgattgt gccggtggcg ctgccg   36

<210> 351
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD482

<400> 351
attctggcgg tggcggcgat tccggtggcg gtgccg   36

<210> 352
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD483

<400> 352
attctggcgg cggcgattat tccggcggcg ctgccg   36

<210> 353
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD484

<400> 353
ctggcggtgg tgctggcggc gccggcgatt gtgccg   36

<210> 354
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD485

<400> 354
gcgattctgg cggcgattgt gccgctggcg gtgccg   36

<210> 355
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD501

<400> 355
gtgattgtgg cgctggcggt gccggcgctg gcgccg   36

<210> 356
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD502

<400> 356
gcgattgtgg cgctggcggt gccggtgctg gcgccg   36

<210> 357
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD503

<400> 357
gcggcgatta ttattgtgct gccggcggcg ctgccg   36

<210> 358
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD504

<400> 358
ctgattgtgg cgctggcggt gccggcgctg gcgccg   36

<210> 359
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD505

<400> 359
gcgattatta ttgtgattgc gccggcggcg gcgccg   36

<210> 360
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD521

<400> 360
ctggcggcgc tgattgtggt gccggcggtg gcgccg   36

<210> 361
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD522

<400> 361
gcgctgctgg tgattgcggt gccggcggtg gcgccg   36

<210> 362
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD524

<400> 362
gcggtggcgc tgattgtggt gccggcgctg gcgccg   36

<210> 363
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD525

<400> 363
gcgctggcga ttgtggtggc gccggtggcg gtgccg   36

<210> 364
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD541

<400> 364
ctgctggcgc tgattattgc gccggcggcg gcgccg   36

<210> 365
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD542

<400> 365
gcgctggcgc tgattattgt gccggcggtg gcgccg   36

<210> 366
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD543

<400> 366
ctgctggcgg cgctgattgc gccggcggcg ctgccg   36

<210> 367
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD544

<400> 367
attgtggcgc tgattgtggc gccggcggcg gtgccg   36

<210> 368
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD545

<400> 368
gtggtgctgg tgctggcggc gccggcggcg gtgccg   36

<210> 369
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD561

<400> 369
gcggcggtgg cgattgtgct gccggcggtg gtgccg   36

<210> 370
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD562

<400> 370
gcgctgattg cggcgattgt gccggcgctg gtgccg   36

<210> 371
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD563

<400> 371
gcgctggcgg tgattgtggt gccggcgctg gcgccg   36

<210> 372
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD564

<400> 372
gtggcgattg cgctgattgt gccggcgctg gcgccg   36

<210> 373
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD565

<400> 373
gtggcgattg tgctggtggc gccggcggtg gcgccg   36
<210> 374
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD582

<400> 374
gtggcggtgg cgctgattgt gccggcgctg gcgccg   36

<210> 375
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD583

<400> 375
gcggtgattc tggcgctggc gccgattgtg gcgccg   36

<210> 376
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD585

<400> 376
gcgctgattg tggcgattgc gccggcgctg gtgccg   36

<210> 377
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD601

<400> 377
gcggcgattc tgattgcggt gccgattgcg gcgccg   36

<210> 378
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD602

<400> 378
gtgattgtgg cgctggcggc gccggtgctg gcgccg   36

<210> 379
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD603

<400> 379
gtgctggtgg cgctggcggc gccggtgatt gcgccg   36

<210> 380
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD604

<400> 380
gtggcgctga ttgcggtggc gccggcggtg gtgccg   36

<210> 381
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD605

<400> 381
gtgattgcgg cggtgctggc gccggtggcg gtgccg   36

<210> 382
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD622

<400> 382
gcgctgattg tgctggcggc gccggtggcg gtgccg   36

<210> 383
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD623

<400> 383
gtggcggcgg cgattgcgct gccggcgatt gtgccg   36

<210> 384
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD625

<400> 384
attctggcgg cggcggcggc gccgctgatt gtgccg   36

<210> 385
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD643

<400> 385
ctggcgctgg tgctggcggc gccggcgatt gtgccg   36

<210> 386
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD645

<400> 386
gcgctggcgg tggtggcgct gccggcgatt gtgccg   36

<210> 387
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD661

<400> 387
gcggcgattc tggcgccgat tgtggcggcg ctgccg   36

<210> 388
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD664

<400> 388
attctgattg cgattgcgat tccggcggcg gcgccg   36

<210> 389
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD665

<400> 389
ctggcgattg tgctggcggc gccggtggcg gtgccg   36

<210> 390
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD666

<400> 390
gcggcgattg cgattattgc gccggcgatt gtgccg   36

<210> 391
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD667

<400> 391
ctggcggtgg cgattgtggc gccggcgctg gtgccg   36

<210> 392
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD683

<400> 392
ctggcgattg tgctggcggc gccggcggtg ctgccg   36

<210> 393
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD684

<400> 393
gcggcgattg tgctggcgct gccggcggtg ctgccg   36

<210> 394
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD685

<400> 394
gcgctgctgg tggcggtgct gccggcggcg ctgccg   36

<210> 395
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD686

<400> 395
gcggcgctgg tggcggtgct gccggtggcg ctgccg   36

<210> 396
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD687

<400> 396
attgtggcgg tggcgctggt gccggcgctg gcgccg   36

<210> 397
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD703

<400> 397
attgtggcgg tggcgctggt gccggcgctg gcgccg   36

<210> 398
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD705

<400> 398
attgtggcgg tggcgctgct gccggcgctg gcgccg   36

<210> 399
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD706

<400> 399
attgtggcgg tggcgctgct gccggcggtg gcgccg   36

<210> 400
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD707

<400> 400
attgtggcgc tggcggtgct gccggcggtg gcgccg   36

<210> 401
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD724

<400> 401
gtggcggtgc tggcggtgct gccggcgctg gcgccg   36

<210> 402
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD725

<400> 402
attgcggtgc tggcggtggc gccggcggtg ctgccg   36
<210> 403
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD726

<400> 403
ctggcggtgg cgattattgc gccggcggtg gcgccg   36

<210> 404
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD727

<400> 404
gtggcgctgg cgattgcgct gccggcggtg ctgccg   36

<210> 405
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD743

<400> 405
gcgattgcga ttgcgctggt gccggtggcg ctgccg   36

<210> 406
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD744

<400> 406
gcggcggtgg tgattgtggc gccggtggcg ctgccg   36

<210> 407
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD746

<400> 407
gcggcgattc tggcgattgt ggcgccgctg gcgccg   36
<210> 408
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD747

<400> 408
gtggcgctgc tggcgattgc gccggcgctg gcgccg   36

<210> 409
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD763

<400> 409
gtggcggtgc tgattgcggt gccggcgctg gcgccg   36

<210> 410
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD764

<400> 410
gcggtggcgc tggcggtgct gccggcggtg gtgccg   36

<210> 411
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD765

<400> 411
gcggtggcgc tggcggtggt gccggcggtg ctgccg   36

<210> 412
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD766

<400> 412
attgtggtga ttgcggtggc gccggcggtg gcgccg   36

<210> 413
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD767

<400> 413
attgtggtgg cggcggtggt gccggcgctg gcgccg   36

<210> 414
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD783

<400> 414
attgtggcgc tggtgccggc ggtggcgatt gcgccg   36

<210> 415
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD784

<400> 415
gtggcggcgc tgccggcggt ggcgctggtg gtgccg   36

<210> 416
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD786

<400> 416
ctggtggcga ttgcgccgct ggcggtgctg gcgccg   36

<210> 417
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD787

<400> 417
gcggtggcgc tggtgccggt gattgtggcg gcgccg   36

<210> 418
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD788

<400> 418
gcgattgcgg tggcgattgc gccggtggcg ctgccg   36

<210> 419
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD803

<400> 419
gcgattgcgc tggcggtgcc ggtgctggcg ctgccg   36

<210> 420
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD805

<400> 420
ctggtgctga ttgcggcggc gccgattgcg ctgccg   36

<210> 421
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD806

<400> 421
ctggtggcgc tggcggtgcc ggcggcggtg ctgccg   36

<210> 422
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD807

<400> 422
gcggtggcgc tggcggtgcc ggcgctggtg ctgccg   36

<210> 423
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD808

<400> 423
ctggtggtgc tggcggcggc gccgctggcg gtgccg   36

<210> 424
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD809

<400> 424
ctgattgtgc tggcggcgcc ggcgctggcg gcgccg   36

<210> 425
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD810

<400> 425
gtgattgtgc tggcggcgcc ggcgctggcg gcgccg   36

<210> 426
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD811

<400> 426
gcggtggtgc tggcggtgcc ggcgctggcg gtgccg   36

<210> 427
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD824

<400> 427
ctgattattg tggcggcggc gccggcggtg gcgccg   36

<210> 428
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD825

<400> 428
attgtggcgg tgattgtggc gccggcggtg gcgccg   36

<210> 429
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD826

<400> 429
ctggtggcgc tggcggcgcc gattattgcg gtgccg   36

<210> 430
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD827

<400> 430
attgcggcgg tgctggcggc gccggcgctg gtgccg   36

<210> 431
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD828

<400> 431
attgcgctgc tggcggcgcc gattattgcg gtgccg   36

<210> 432
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD829

<400> 432
gcggcgctgg cgctggtggc gccggtgatt gtgccg   36

<210> 433
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD830

<400> 433
attgcgctgg tggcggcgcc ggtggcgctg gtgccg   36

<210> 434
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD831

<400> 434
attattgtgg cggtggcgcc ggcggcgatt gtgccg   36

<210> 435
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD832

<400> 435
gcggtggcgg cgattgtgcc ggtgattgtg gcgccg   36

<210> 436
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD843

<400> 436
gcggtgctgg tgctggtggc gccggcggcg gcgccg   36
<210> 437
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD844

<400> 437
gtggtggcgc tgctggcgcc gctgattgcg gcgccg   36

<210> 438
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD845

<400> 438
gcggcggtgg tgattgcgcc gctgctggcg gtgccg   36

<210> 439
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD846

<400> 439
attgcggtgg cggtggcggc gccgctgctg gtgccg   36

<210> 440
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD847

<400> 440
ctggtggcga ttgtggtgct gccggcggtg gcgccg   36

<210> 441
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD848

<400> 441
gcggtggcga ttgtggtgct gccggcggtg gcgccg   36

<210> 442
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD849

<400> 442
gcggtgattc tgctggcgcc gctgattgcg gcgccg   36

<210> 443
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD850

<400> 443
ctggtgattg cgctggcggc gccggtggcg ctgccg   36

<210> 444
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD851

<400> 444
gtgctggcgg tggtgctgcc ggcggtggcg ctgccg   36

<210> 445
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD852

<400> 445
gtgctggcgg tggcggcgcc ggcggtgctg ctgccg   36

<210> 446
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD863

<400> 446
gcggcggtgg tgctgctgcc gattattgcg gcgccg   36

<210> 447
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD864

<400> 447
gcgctgctgg tgattgcgcc ggcgattgcg gtgccg   36

<210> 448
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD865

<400> 448
gcggtgctgg tgattgcggt gccggcgatt gcgccg   36

<210> 449
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD867

<400> 449
gcgctgctgg tggtgattgc gccgctggcg gcgccg   36

<210> 450
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD868

<400> 450
gtgctggtgg cggcgattct gccggcggcg attccg   36

<210> 451
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD870

<400> 451
gtgctggtgg cggcggtgct gccgattgcg gcgccg   36

<210> 452
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD872

<400> 452
gtgctggcgg cggcggtgct gccgctggtg gtgccg   36

<210> 453
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD875

<400> 453
gcgattgcga ttgtggtgcc ggcggtggcg gtgccg   36

<210> 454
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD877

<400> 454
gtggcgatta ttgcggtgcc ggcggtggtg gcgccg   36

<210> 455
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD878

<400> 455
attgtggcgc tggtggcgcc ggcggcggtg gtgccg   36

<210> 456
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD879

<400> 456
gcggcgattg tgctgctgcc ggcggtggtg gtgccg   36

<210> 457
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD881

<400> 457
gcggcgctga ttgtggtgcc ggcggtggcg gtgccg   36

<210> 458
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD882

<400> 458
gcgattgcgc tggtggtgcc ggcggtggcg gtgccg   36

<210> 459
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD883

<400> 459
ctggcgattg tgccggcggc gattgcggcg ctgccg   36

<210> 460
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD885

<400> 460
ctggtggcga ttgcgccggc ggtggcggtg ctgccg   36

<210> 461
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD887

<400> 461
gtgctggcgg tggcgccggc ggtggcggtg ctgccg   36

<210> 462
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD888

<400> 462
attctggcgg tggtggcgat tccggcggcg gcgccg   36

<210> 463
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD889

<400> 463
attctggtgg cggcggcgcc gattgcggcg ctgccg   36

<210> 464
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD891

<400> 464
attctggcgg tggcggcgat tccggcggcg ctgccg   36

<210> 465
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD893

<400> 465
gtgattgcga ttccggcgat tctggcggcg gcgccg   36
<210> 466
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD895

<400> 466
gcgattatta ttgtggtgcc ggcgattgcg gcgccg   36

<210> 467
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD896

<400> 467
gcgattctga ttgtggtggc gccgattgcg gcgccg   36

<210> 468
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD897

<400> 468
gcggtgattg tgccggtggc gattattgcg gcgccg   36

<210> 469
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD899

<400> 469
gcggtggtga ttgcgctgcc ggcggtggtg gcgccg   36

<210> 470
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD900

<400> 470
gcgctggtgg cggtgattgc gccggtggtg gcgccg   36
<210> 471
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD901

<400> 471
gcgctggtgg cggtgctgcc ggcggtggcg gtgccg   36

<210> 472
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD902

<400> 472
gcgctggtgg cgccgctgct ggcggtggcg gtgccg   36

<210> 473
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD904

<400> 473
gcggtgctgg cggtggtggc gccggtggtg gcgccg   36

<210> 474
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD905

<400> 474
gcggtgattg cggtggcgcc gctggtggtg gcgccg   36

<210> 475
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD906

<400> 475
gcggtgattg cgctggcgcc ggtggtggtg gcgccg   36

<210> 476
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD907

<400> 476
gtggcgattg cgctggcgcc ggtggtggtg gcgccg   36

<210> 477
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD908

<400> 477
gtggcgctgg cgctggcgcc ggtggtggtg gcgccg   36

<210> 478
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD910

<400> 478
gtggcggcgc tgctgccggc ggtggtggtg gcgccg   36

<210> 479
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD911

<400> 479
gtggcgctgg cgctgccggc ggtggtggtg gcgccg   36

<210> 480
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD912

<400> 480
gtggcgctgc tggcgccggc ggtggtggtg gcgccg   36

<210> 481
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD1 5'-primer

<400> 481

<210> 482
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD2 5'-primer

<400> 482

<210> 483
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD3 5'-primer

<400> 483

<210> 484
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD4 5'-primer

<400> 484

<210> 485
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD5 5'-primer

<400> 485

<210> 486
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD6 5'-primer

<400> 486

<210> 487
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD9 5'-primer

<400> 487

<210> 488
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD11 5'-primer

<400> 488

<210> 489
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD12 5'-primer

<400> 489

<210> 490
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD13 5'-primer

<400> 490

<210> 491
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD16 5'-primer

<400> 491

<210> 492
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD17 5'-primer

<400> 492

<210> 493
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD18 5'-primer

<400> 493

<210> 494
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD19 5'-primer

<400> 494

<210> 495
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD20 5'-primer

<400> 495

<210> 496
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD21 5'-primer

<400> 496

<210> 497
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD22 5'-primer

<400> 497

<210> 498
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD23 5'-primer

<400> 498

<210> 499
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD24 5'-primer

<400> 499

<210> 500
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD25 5'-primer

<400> 500

<210> 501
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD26 5'-primer

<400> 501

<210> 502
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD27 5'-primer

<400> 502

<210> 503
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD28 5'-primer

<400> 503

<210> 504
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD29 5'-primer

<400> 504

<210> 505
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD30 5'-primer

<400> 505

<210> 506
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD33 5'-primer

<400> 506

<210> 507
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD37 5'-primer

<400> 507

<210> 508
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD38 5'-primer

<400> 508

<210> 509
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD39 5'-primer

<400> 509

<210> 510
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD40 5'-primer

<400> 510

<210> 511
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD42 5'-primer

<400> 511

<210> 512
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD43 5'-primer

<400> 512

<210> 513
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD44 5'-primer

<400> 513

<210> 514
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD49 5'-primer

<400> 514

<210> 515
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD54 5'-primer

<400> 515

<210> 516
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD57 5'-primer

<400> 516

<210> 517
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD59 5'-primer

<400> 517

<210> 518
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD61 5'-primer

<400> 518

<210> 519
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD62 5'-primer

<400> 519

<210> 520
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD63 5'-primer

<400> 520

<210> 521
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD64 5'-primer

<400> 521

<210> 522
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD65 5'-primer

<400> 522

<210> 523
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD66 5'-primer

<400> 523

<210> 524
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD67 5'-primer

<400> 524

<210> 525
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD68 5'-primer

<400> 525

<210> 526
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD69 5'-primer

<400> 526

<210> 527
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD71 5'-primer

<400> 527

<210> 528
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD77 5'-primer

<400> 528

<210> 529
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD81 5'-primer

<400> 529

<210> 530
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD82 5'-primer

<400> 530

<210> 531
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD83 5'-primer

<400> 531

<210> 532
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD84 5'-primer

<400> 532

<210> 533
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD85 5'-primer

<400> 533

<210> 534
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD97 5'-primer

<400> 534

<210> 535
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD101 5'-primer

<400> 535

<210> 536
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD102 5'-primer

<400> 536

<210> 537
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD103 5'-primer

<400> 537

<210> 538
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD104 5'-primer

<400> 538

<210> 539
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD105 5'-primer

<400> 539

<210> 540
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD113 5'-primer

<400> 540

<210> 541
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD121 5'-primer

<400> 541

<210> 542
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD123 5'-primer

<400> 542

<210> 543
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD124 5'-primer

<400> 543

<210> 544
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD131 5'-primer

<400> 544

<210> 545
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD138 5'-primer

<400> 545

<210> 546
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD139 5'-primer

<400> 546

<210> 547
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD141 5'-primer

<400> 547

<210> 548
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD142 5'-primer

<400> 548

<210> 549
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD143 5'-primer

<400> 549

<210> 550
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD144 5'-primer

<400> 550

<210> 551
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD145 5'-primer

<400> 551

<210> 552
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD152 5'-primer

<400> 552

<210> 553
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD159 5'-primer

<400> 553

<210> 554
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD161 5'-primer

<400> 554

<210> 555
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD162 5'-primer

<400> 555

<210> 556
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD163 5'-primer

<400> 556

<210> 557
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD164 5'-primer

<400> 557

<210> 558
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD165 5'-primer

<400> 558

<210> 559
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD167 5'-primer

<400> 559

<210> 560
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD169 5'-primer

<400> 560

<210> 561
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD182 5'-primer

<400> 561

<210> 562
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD183 5'-primer

<400> 562

<210> 563
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD184 5'-primer

<400> 563

<210> 564
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD185 5'-primer

<400> 564

<210> 565
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD189 5'-primer

<400> 565

<210> 566
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD190 5'-primer

<400> 566

<210> 567
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD201 5'-primer

<400> 567

<210> 568
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD204 5'-primer

<400> 568

<210> 569
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD205 5'-primer

<400> 569

<210> 570
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD210 5'-primer

<400> 570

<210> 571
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD214 5'-primer

<400> 571

<210> 572
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD221 5'-primer

<400> 572

<210> 573
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD222 5'-primer

<400> 573

<210> 574
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD223 5'-primer

<400> 574

<210> 575
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD224 5'-primer

<400> 575

<210> 576
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD225 5'-primer

<400> 576

<210> 577
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD226 5'-primer

<400> 577

<210> 578
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD227 5'-primer

<400> 578

<210> 579
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD241 5'-primer

<400> 579

<210> 580
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD242 5'-primer

<400> 580

<210> 581
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD243 5'-primer

<400> 581

<210> 582
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD245 5'-primer

<400> 582

<210> 583
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD246 5'-primer

<400> 583

<210> 584
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD248 5'-primer

<400> 584

<210> 585
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD261 5'-primer

<400> 585

<210> 586
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD262 5'-primer

<400> 586

<210> 587
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD263 5'-primer

<400> 587

<210> 588
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD264 5'-primer

<400> 588

<210> 589
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD265 5'-primer

<400> 589

<210> 590
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD281 5'-primer

<400> 590

<210> 591
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD282 5'-primer

<400> 591

<210> 592
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD283 5'-primer

<400> 592

<210> 593
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD284 5'-primer

<400> 593

<210> 594
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD285 5'-primer

<400> 594

<210> 595
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD301 5'-primer

<400> 595

<210> 596
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD302 5'-primer

<400> 596

<210> 597
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD304 5'-primer

<400> 597

<210> 598
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD305 5'-primer

<400> 598

<210> 599
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD321 5'-primer

<400> 599

<210> 600
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD322 5'-primer

<400> 600

<210> 601
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD323 5'-primer

<400> 601

<210> 602
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD324 5'-primer

<400> 602

<210> 603
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD325 5'-primer

<400> 603

<210> 604
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD329 5'-primer

<400> 604

<210> 605
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD331 5'-primer

<400> 605

<210> 606
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD341 5'-primer

<400> 606

<210> 607
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD342 5'-primer

<400> 607

<210> 608
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD343 5'-primer

<400> 608

<210> 609
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD345 5'-primer

<400> 609

<210> 610
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD349 5'-primer

<400> 610

<210> 611
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD350 5'-primer

<400> 611

<210> 612
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD361 5'-primer

<400> 612

<210> 613
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD363 5'-primer

<400> 613

<210> 614
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD364 5'-primer

<400> 614

<210> 615
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD365 5'-primer

<400> 615

<210> 616
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD381 5'-primer

<400> 616

<210> 617
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD382 5'-primer

<400> 617

<210> 618
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD383 5'-primer

<400> 618

<210> 619
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD384 5'-primer

<400> 619

<210> 620
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD385 5'-primer

<400> 620

<210> 621
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD390 5'-primer

<400> 621

<210> 622
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD401 5'-primer

<400> 622

<210> 623
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD402 5'-primer

<400> 623

<210> 624
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD403 5'-primer

<400> 624

<210> 625
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD404 5'-primer

<400> 625

<210> 626
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD405 5'-primer

<400> 626

<210> 627
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD421 5'-primer

<400> 627

<210> 628
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD422 5'-primer

<400> 628

<210> 629
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD424 5'-primer

<400> 629

<210> 630
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD425 5'-primer

<400> 630

<210> 631
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD426 5'-primer

<400> 631

<210> 632
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD436 5'-primer

<400> 632

<210> 633
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD442 5'-primer

<400> 633

<210> 634
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD443 5'-primer

<400> 634

<210> 635
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD444 5'-primer

<400> 635

<210> 636
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD445 5'-primer

<400> 636

<210> 637
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD461 5'-primer

<400> 637

<210> 638
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD462 5'-primer

<400> 638

<210> 639
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD463 5'-primer

<400> 639

<210> 640
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD464 5'-primer

<400> 640

<210> 641
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD465 5'-primer

<400> 641

<210> 642
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD466 5'-primer

<400> 642

<210> 643
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD481 5'-primer

<400> 643

<210> 644
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD482 5'-primer

<400> 644

<210> 645
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD483 5'-primer

<400> 645

<210> 646
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD484 5'-primer

<400> 646

<210> 647
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD485 5'-primer

<400> 647

<210> 648
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD501 5'-primer

<400> 648

<210> 649
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD502 5'-primer

<400> 649

<210> 650
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD503 5'-primer

<400> 650

<210> 651
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD504 5'-primer

<400> 651

<210> 652
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD505 5'-primer

<400> 652

<210> 653
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD521 5'-primer

<400> 653

<210> 654
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD522 5'-primer

<400> 654

<210> 655
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD524 5'-primer

<400> 655

<210> 656
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD525 5'-primer

<400> 656

<210> 657
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD527 5'-primer

<400> 657

<210> 658
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD541 5'-primer

<400> 658

<210> 659
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD542 5'-primer

<400> 659

<210> 660
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD543 5'-primer

<400> 660

<210> 661
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD544 5'-primer

<400> 661

<210> 662
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD545 5'-primer

<400> 662

<210> 663
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD561 5'-primer

<400> 663

<210> 664
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD562 5'-primer

<400> 664

<210> 665
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD563 5'-primer

<400> 665

<210> 666
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD564 5'-primer

<400> 666

<210> 667
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD565 5'-primer

<400> 667

<210> 668
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD577 5'-primer

<400> 668

<210> 669
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD582 5'-primer

<400> 669

<210> 670
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD583 5'-primer

<400> 670

<210> 671
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD585 5'-primer

<400> 671

<210> 672
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD601 5'-primer

<400> 672

<210> 673
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD602 5'-primer

<400> 673

<210> 674
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD603 5'-primer

<400> 674

<210> 675
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD604 5'-primer

<400> 675

<210> 676
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD605 5'-primer

<400> 676

<210> 677
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD606 5'-primer

<400> 677

<210> 678
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD622 5'-primer

<400> 678

<210> 679
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD623 5'-primer

<400> 679

<210> 680
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD625 5'-primer

<400> 680

<210> 681
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD635 5'-primer

<400> 681

<210> 682
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD643 5'-primer

<400> 682

<210> 683
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD645 5'-primer

<400> 683

<210> 684
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD661 5'-primer

<400> 684

<210> 685
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD664 5'-primer

<400> 685

<210> 686
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD665 5'-primer

<400> 686

<210> 687
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD666 5'-primer

<400> 687

<210> 688
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD667 5'-primer

<400> 688

<210> 689
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD676 5'-primer

<400> 689

<210> 690
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD683 5'-primer

<400> 690

<210> 691
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD684 5'-primer

<400> 691

<210> 692
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD685 5'-primer

<400> 692

<210> 693
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD686 5'-primer

<400> 693

<210> 694
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD687 5'-primer

<400> 694

<210> 695
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD692 5'-primer

<400> 695

<210> 696
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD693 5'-primer

<400> 696

<210> 697
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD700 5'-primer

<400> 697

<210> 698
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD703 5'-primer

<400> 698

<210> 699
<211> 68
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD705 5'-primer

<400> 699

<210> 700
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD706 5'-primer

<400> 700

<210> 701
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD707 5'-primer

<400> 701

<210> 702
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD724 5'-primer

<400> 702