(19)
(11)EP 3 334 756 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
17.06.2020 Bulletin 2020/25

(21)Application number: 16835419.9

(22)Date of filing:  09.08.2016
(51)Int. Cl.: 
C07K 14/47  (2006.01)
C12N 5/074  (2010.01)
C07K 7/08  (2006.01)
C12N 15/85  (2006.01)
(86)International application number:
PCT/KR2016/008760
(87)International publication number:
WO 2017/026779 (16.02.2017 Gazette  2017/07)

(54)

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

VERBESSERTES REKOMBINANTES PROTEIN MIT ZELLDURCHLÄSSIGEM CRE (ICP-CRE) UND VERWENDUNG DAVON

PROTÉINE RECOMBINÉE CRE À PERMÉABILITÉ CELLULAIRE AMÉLIORÉE (ICP-CRE) ET SON UTILISATION


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 10.08.2015 US 201562202990 P

(43)Date of publication of application:
20.06.2018 Bulletin 2018/25

(73)Proprietor: Cellivery Therapeutics, Inc.
Seoul 03929 (KR)

(72)Inventor:
  • JO, Daewoong
    Brentwood, Tennessee 37207 (US)

(74)Representative: Isarpatent 
Patent- und Rechtsanwälte Behnisch Barth Charles Hassa Peckmann & Partner mbB Friedrichstrasse 31
80801 München
80801 München (DE)


(56)References cited: : 
WO-A1-2016/028036
WO-A2-03/076561
US-A1- 2006 222 657
WO-A2-02/20737
US-A- 5 646 016
US-A1- 2010 197 598
  
  • JO D ET AL: "Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase", NATURE BIOTECHNOLOGY, GALE GROUP INC, vol. 19, no. 10, 1 October 2001 (2001-10-01), pages 929-933, XP002277910, ISSN: 1087-0156, DOI: 10.1038/NBT1001-929
  • LIN Y ET AL: "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, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 270, no. 24, 16 June 1995 (1995-06-16), pages 14255-14258, XP002050723, ISSN: 0021-9258, DOI: 10.1074/JBC.270.24.14255
  • DATABASE NUCLEOTIDE [Online] 27 July 2013 XP055463078 Retrieved from NCBI Database accession no. KC845567
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


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. 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. 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. 3. Lapenna S, Giordano A., Cell cycle kinases as therapeutic targets for cancer, Nat Rev Drug Discov. 2009;8:547-66.
  4. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 16. Sauer B. Inducible gene targeting in mice using the Cre/lox system. Methods 1998;14(4):381-92.
  17. 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. 18. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science 1995;269(5229): 1427-9.
  19. 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. 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. 21. Chen CM, Behringer RR. CREating breakthroughs. Nature Biotechnology 2001;19(10):921-2.
  22. 22. Kolb AF, Siddell SG. Genomic targeting with an MBP-Cre fusion protein. Gene 1996; 183(1-2):53-60.
  23. 23. Baubonis W, Sauer B. Genomic targeting with purified Cre recombinase. Nucleic Acids Research 1993;21(9):2025-9.
  24. 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. 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. 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. 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. 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:
  1. a.Amino acid length: 9 to 13
  2. b.Bending potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end (12') of the sequence.
  3. c.Rigidity/Flexibility: Instability Index (II): 40 to 60
  4. d.Structural Feature: Aliphatic Index (AI): 180 to 220
  5. e.Hydropathy: GRAVY: 2.1 to 2.6
  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:
  1. a.Amino Acid Length: 9 to 13
  2. b.Bending Potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end of the sequence.
  3. c.Rigidity/Flexibility: Instability Index (II): 40 to 60
  4. d.Structural Feature: Aliphatic Index (AI): 180 to 220
  5. e.Hydropathy: GRAVY: 2.1 to 2.6.
  6. f.Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)
  7. 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:
  1. a.Amino Acid Length: 9 to 13
  2. b.Bending Potential: Proline (P) positioned in the middle (5', 6', 7' or 8') and at the end of the sequence.
  3. c.Rigidity/Flexibility: Instability Index (II): 40 to 60
  4. d.Structural Feature: Aliphatic Index (AI): 180 to 220
  5. e.Hydropathy: GRAVY: 2.1 to 2.6.
  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:
  1. a.Amino Acid Sequence: 12
  2. b.Bending Potential: Proline (P) is positioned in the middle (5', 6', 7' or 8') and at the end (12') of the sequence.
  3. c.Rigidity/Flexibility: Instability Index (II): 41.3 to 57.3
  4. d.Structural Feature: Aliphatic Index (AI): 187.5 to 220
  5. e.Hydropathy: GRAVY: 2.2 to 2.6.
  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:
  1. (a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;
  2. (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
  3. (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:
  1. (a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;
  2. (b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence;
  3. (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
  4. (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:
  1. (a) being preferably composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;
  2. (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
  3. (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:

  1. (a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;
  2. (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;
  3. (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
  4. (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. 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. 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. 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. 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. 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 SOCS3f/f Conditional Knockout Mice according to example <14-3>. SOCS3f/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 SOCS3f/f Conditional Knockout Mice according to example <14-3>. SOCS3f/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 SOCS3f/f Conditional Knockout Mice according to example <14-3>. SOCS3f/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 SOCS3f/f Conditional Knockout Mice according to example <14-3>. SOCS3f/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 SOCS3f/f Conditional Knockout Mice Treated by Local Administration according to example <14-3>. SOCS3f/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 ROSAnT-nG Mice according to example <14-4>. ROSAnT-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]
#PepidesOriginProteinRef.
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
TitleJournalYearVolIssuePage
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]
#PeptidesSequenceLengthMolecular WeightpIBending PotentialRigidity/Flexibility (Instability Index : II)Structural Feature (Aliphatic Index : AI)Hydropathy (GRAVY)Residue StructureA/a CompositionSecondary StructureCargoRef.
AVLIPG
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]
#PeptidesSequenceLengthMolecular WeightpIBending PotentialRigidity/Flexibility (Instability Index : II)Structural Feature (Aliphatic Index : AI)Hydropathy (GRAVY)Residue StructureA/a CompositionSecondary StructureCargoRef.
AVLIPG
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]
#PeptidesSequenceLengthMolecular WeightpIBending PotentialRigidity/Flexibility (Instability Index : II)Structural Feature (Aliphatic Index : AI)Hydropathy (GRAVY)Residue StructureA/a CompositionSecondary StructureCargoRef.
AVLIPG
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 FactorSelected CPPsNewly Designed CPPs
RangeRange
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 NumberaMTDSequencesLengthRigidity/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 NumberaMTDSequencesLengthRigidity/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 NumberaMTDSequencesLengthRigidity/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 NumberaMTDSequencesLengthRigidity/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 NumberaMTDSequencesLengthRigidity/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 NumberaMTDSequencesLengthRigidity/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]
GrouprPeptide IDSequencesLengthProline 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]
GrouprPeptide IDSequencesLengthProline 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]
GrouprPeptide IDSequencesLengthProline 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]
GrouprPeptide IDSequencesLengthProline 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]
GrouprPeptide IDSequencesLengthProline 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]
GrouprPeptide IDSequencesLengthProline 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 NumberaMTDSequencesLengthProline PositionRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)
(PP)(II)(AI)(GRAVY)ABC
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 NumberaMTDSequencesLengthProline PositionRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)
(PP)(II)(AI)(GRAVY)ABC
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 NumberaMTDSequencesLengthProline PositionRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)
(PP)(II)(AI)(GRAVY)ABC
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 NumberaMTDSequencesLengthProline PositionRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)
(PP)(II)(AI)(GRAVY)ABC
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 NumberaMTDSequencesLengthProline PositionRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)
(PP)(II)(AI)(GRAVY)ABC
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 rP38MTM12MTD85
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]
NumberIDSequenceLengthProline 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 PotentialRigidity/FlexibilitySturctural FeatureHydropathyRelative Ratio (Fold)Amino Acid Composition
(II)(AI)(GRAVY)ABCAVIL
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 FactorNewly Designed CPPsAnalysis of Experimental Results
RangeRange
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 NOaMTD IDAmino 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]
SDGenbank IDOriginProtein (kDa)pIInstability 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 SOCS3f/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 ROSAnT-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]
CargoRecombinant Protein5' Primers (5' → 3')3' Primer (5' → 3')
Cre HNC

 


 
HNMCB

 


 
HNMAC

 


 
HNMACB

 
[Table 39]
CargoSDRecombinant Protein5' 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]
Gene5' 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
RankaMTDYield (mg/L) RankaMTD RankaMTD
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] SOCS3f/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 SOCS3f/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, SOCS3f/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 SOCS3f/f mice by portal vein injection with the iCP-Cre recombinant protein, and inhibited in the kidney of the SOCS3f/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] ROSAnT-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 ROSAnT-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

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<223> Amino acid Sequence of aMTD243

<400> 58

<210> 59
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD245

<400> 59

<210> 60
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD261

<400> 60

<210> 61
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD262

<400> 61

<210> 62
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD263

<400> 62

<210> 63
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD264

<400> 63

<210> 64
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD265

<400> 64

<210> 65
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD281

<400> 65

<210> 66
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD282

<400> 66

<210> 67
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD283

<400> 67

<210> 68
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD284

<400> 68

<210> 69
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD285

<400> 69

<210> 70
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD301

<400> 70

<210> 71
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD302

<400> 71

<210> 72
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD304

<400> 72

<210> 73
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD305

<400> 73

<210> 74
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD321

<400> 74

<210> 75
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD322

<400> 75

<210> 76
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD323

<400> 76

<210> 77
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD324

<400> 77

<210> 78
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD325

<400> 78

<210> 79
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD341

<400> 79

<210> 80
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD342

<400> 80

<210> 81
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD343

<400> 81

<210> 82
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD345

<400> 82

<210> 83
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD361

<400> 83

<210> 84
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD363

<400> 84

<210> 85
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD364

<400> 85

<210> 86
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD365

<400> 86

<210> 87
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD381

<400> 87

<210> 88
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD382

<400> 88

<210> 89
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD383

<400> 89

<210> 90
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD384

<400> 90

<210> 91
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD385

<400> 91

<210> 92
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD401

<400> 92

<210> 93
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD402

<400> 93

<210> 94
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD403

<400> 94

<210> 95
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD404

<400> 95

<210> 96
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD405

<400> 96

<210> 97
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD421

<400> 97

<210> 98
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD422

<400> 98

<210> 99
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD424

<400> 99

<210> 100
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD425

<400> 100

<210> 101
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD442

<400> 101

<210> 102
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD443

<400> 102

<210> 103
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD444

<400> 103

<210> 104
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD445

<400> 104

<210> 105
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD461

<400> 105

<210> 106
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD462

<400> 106

<210> 107
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD463

<400> 107

<210> 108
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD464

<400> 108

<210> 109
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD465

<400> 109

<210> 110
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD481

<400> 110

<210> 111
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD482

<400> 111

<210> 112
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD483

<400> 112

<210> 113
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD484

<400> 113

<210> 114
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD485

<400> 114

<210> 115
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD501

<400> 115

<210> 116
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD502

<400> 116

<210> 117
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD503

<400> 117

<210> 118
<211> 12
<212> PRT
<213> Artificial Sequence

<220>
<223> Amino acid Sequence of aMTD504

<400> 118

<210> 119
<211> 12
<212> PRT
<213> Artificial Sequence

<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

<210> 703
<211> 69
<212> DNA
<213> Artificial Sequence

<220>
<223> cDNA Sequence of aMTD725 5'-primer

<400> 703