| (11) | EP 3 286 314 B1 |
(12) | EUROPEAN PATENT SPECIFICATION |
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(54) | COW ANTIBODY SCAFFOLD POLYPEPTIDE METHOD AND COMPOSITION KUH-ANTIKÖRPERGERÜSTPOLYPEPTIDVERFAHREN UND -ZUSAMMENSETZUNG PROCÉDÉ ET COMPOSITION DE POLYPEPTIDE D'ÉCHAFAUDAGE D'ANTICORPS BOVIN |
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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). |
TECHNICAL FIELD
BACKGROUND
SUMMARY
1. A method of selecting a peptide of interest, the method comprising the steps of
2. The method of clause 1 wherein the step of preparing the peptide library comprises the step of in vitro transcription of a DNA library to form an mRNA library.
3. The method of clause 2 further comprising the step of digesting the DNA library with DNase.
4. The method of clause 2 or 3 further comprising the step of conjugating the mRNA from the mRNA library to a puromycin oligonucleotide linker.
5. The method of any one of clauses 2 to 4 further comprising the step of translating in vitro the mRNA from the mRNA library to form mRNA-cow antibody scaffold polypeptide fusion conjugates comprising the cow antibody scaffold polypeptide wherein the cow antibody scaffold polypeptide comprises a purification tag.
6. The method of clause 5 further comprising the step of purifying the mRNA-cow antibody scaffold polypeptide fusion conjugates using the purification tag.
7. The method of clause 5 or 6 further comprising the step of reverse transcribing the mRNA from the mRNA library to form mRNA-DNA duplexes of the mRNA-cow antibody scaffold polypeptide fusion conjugates.
8. The method of clause 7 wherein the step of selecting comprises contacting the mRNA-DNA duplexes of the mRNA-cow antibody scaffold polypeptide fusion conjugates with the target molecule.
9. The method of clause 8 further comprising the step of regenerating the DNA from the mRNA-DNA duplexes of the mRNA-cow antibody scaffold polypeptide fusion conjugates.
10. The method of any one of clauses 2 to 9 wherein the DNA library is a cDNA library.
11. The method of any one of clauses 2 to 10 wherein the in vitro transcription is performed using T7 RNA polymerase.
12. The method of any one of clauses 4 to 11 wherein the puromycin oligonucleotide linker is linked to the mRNA at the 3' end of the mRNA.
13. The method of any one of clauses 5 to 12 wherein the purification tag is a FLAG tag.
14. The method of any one of clauses 2 to 13 wherein members of the DNA library comprise an RNA polymerase promoter sequence, an enhancer sequence, and a purification tag sequence.
15. The method of any one of clauses 5 to 13 wherein the purification tag is a C-terminal tag.
18. The cow antibody scaffold polypeptide has to comprise the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO: 3) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
19. The method of any one of clauses 18 wherein X* comprises a natural amino acid.
20. The method of any one of clauses 18 wherein X* comprises a non-natural amino acid.
21. The method of any one of clauses 18 to 20 wherein c is an integer from 1 to 40.
22. The method of any one of clauses 18 to 20 wherein c is an integer from 1 to 10.
23. The method of any one of clauses above further comprising the steps of:
wherein each R1, R2, R3 and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and a N-terminal protecting group;
R7 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R8 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R9 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain, and a non-natural amino acid side chain;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
each L' and L" is independently an optional bivalent linking group or a bond;
b is an integer from 0 to 50;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 6;
r is 0 or 1;
s is an integer from 0 to 100;
t is 0 or 1;
u is 0 or 1;
v is an integer from 0 to 100;
w is 0 or 1; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface; and *** is a point of connection to the rest of the functionalized peptide;
the method comprising the step of reacting a functionalized peptide of formula II under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L', L", *, and *** are as defined for formula I;
R10 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R11 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R12 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q' is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z';
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
g is an integer from 0 to 50; and
y is 0 or 1;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
24. The method of clause 23, wherein Z comprises a moiety selected from the group consisting of an amide bond,
and
wherein d is an integer from 0 to 6, e is an integer from 0 to 6, and f is an integer from 0 to 6, and ** is a point of connection to the rest of the cyclic peptide.
25. The method of clause 23 or 24, wherein Z comprises a peptide bond, Z" comprises an N-terminal protecting group, t is 0, u is 0, and y is 0.
26. The method of clause 25, further comprising removing Z" from the rest of the functionalized peptide to cause the peptide bond to form.
27. The method of clause 23 or 24, wherein Q and X are bonds to Z, Z comprises ** -S-S- **, X' is a bond to Z", Q' is a bond to Z', Z' and Z" comprise cysteine side chains, t is 1, u is 0, v is 0, w is 1, and y is 0.
28. The method of clause 27, further comprising subjecting the functionalized peptide to oxidative conditions to cause ** -S-S- ** to form.
29. The method of clause 23 or 24, wherein X and Y are bonds to Z, Z comprises ** -S-S- **, X' is a bond to Z", Y' is a bond to Z', Z' and Z" comprise cysteine side chains, t is 1, u is 1, and y is 1.
30. The method of clause 29, further comprising subjecting the functionalized peptide to oxidative conditions to cause ** -S-S- ** to form.
31. The method of clause 23 or 24, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an azide, d is 1, u is 0, v is 0, w is 1, and y is 0.
32. The method of clause 31, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
33. The method of clause 23 or 24, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, d is 1, u is 1, and y is 1.
34. The method of clause 33, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
35. The method of clause 23 or 24, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an azide, e is 1, u is 0, v is 0, w is 1, and y is 0.
36. The method of clause 35, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
37. The method of clause 23 or 24, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, e is 1, u is 1, and y is 1.
38. The method of clause 37, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
39. The method of clause 23 or 24, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an amine, f is 1, u is 0, v is 0, w is 1, and y is 0.
40. The method of clause 39, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
41. The method of clause 23 or 24, wherein Y is a bond to Z, Z comprises
Z" comprises an amine, Y' is a bond to Z', Z' comprises
f is 1, u is 1, and y is 1.
42. The method of clause 41, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
43. The method of clause 23 or 24, wherein R4, R10, and R11 are defined such that the functionalized peptide comprises a butelase 1 recognition sequence, Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' is an asparagine or aspartic acid side chain, u is 1, and y is 1.
44. The method of clause 43, further comprising contacting the functionalized peptide with butelase 1 to cause
to form.
45. The method of clause 23 or 24, wherein Q and X are bonds to Z, Z comprises
Q' is a bond to Z', X' is a bond to Z", Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain, t is 1, u is 0, v is 0, w is 1, and y is 0.
46. The method of clause 45, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
47. The method of clause 23 or 24, wherein X and Y are bonds to Z, Z comprises
X' is a bond to Z", Y' is a bond to Z', Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain, t is 1, u is 1, and y is 1.
48. The method of clause 47, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
49. The method of any one of clauses 23 to 48, wherein each L' and L" is independently of the formula V
wherein each R13 and R13' is independently selected from the group consisting of H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 7-membered heteroaryl, -OR14, -OC(O)R14, -NR14R14', -NR14C(O)R15, -C(O)R14, -C(O)OR14, and -C(O)NR14R14', wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl is independently optionally substituted by halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR16; each R14, R14', R15, and R16 is independently selected from the group consisting of H, D, hydroxyl, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl; and h is an integer from 1 to 10; or the formula VI or VII
wherein j is an integer from 0 to 30.
50. The method of clause 49, wherein each R13 and R13' is hydrogen.
51. The method of clause 49 or 50, wherein L' is present, h is 5, m is 0, n is 1, and p is 1.
52. The method of clause 49, wherein at least one of L' and L" is of the formula VI or VII
wherein j is 7.
53. The method of any one of clauses 23 to 52, wherein the N-terminal protecting group is a photoprotecting group.
54. The method of any one of clauses 23 to 53, wherein the N-terminal protecting group is 2-(2-nitrophenyl)propyloxycarbonyl.
55. The method of any one of clauses 1 to 22 further comprising the step of synthesizing the cow antibody scaffold polypeptide on one or more peptide microarrays wherein the cow antibody scaffold polypeptide comprises the peptide of interest.
56. The method of clause 55 comprising the steps of:
wherein each R1, R2, R3, and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently a natural amino acid side chain or a non-natural amino acid side chain selected such that
can form a beta-sheet;
each R7 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and an N-terminal protecting group;
R8 is selected from the group consisting of hydrogen, an N-terminal capping group, and a protecting group;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
L' is an optional bivalent linking group or a bond;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 50;
r is an integer from 0 to 50;
s is an integer from 0 to 50;
t is an integer from 0 to 50;
u is an integer from 0 to 50; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface;
the method comprising the step of reacting a functionalized peptide of formula IX under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, R7, and R8, m, n, p, q, r, s, t, u, L' and * are as defined for formula VIII;
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z'; and
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
57. The method of clause 56, wherein Z comprises a moiety selected from the group consisting of an amide bond,
and
wherein d is an integer from 0 to 6, e is an integer from 0 to 6, and f is an integer from 0 to 6, and ** is a point of connection to the rest of the cyclic peptide.
58. The method of clause 56 or 57, wherein X and Y are bonds to Z, Z comprises ** -S-S- **, X' is a bond to Z", Y' is a bond to Z', and Z' and Z" comprise cysteine side chains.
59. The method of clause 58, further comprising subjecting the functionalized peptide to oxidative conditions to cause ** -S-S- ** to form.
60. The method of claim 56 or 57, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, and d is 1.
61. The method of clause 60, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
62. The method of clause 56 or 57, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, and e is 1.
63. The method of clause 62, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
64. The method of clause 56 or 57, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an amine, and f is 1.
65. The method of clause 64, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
66. The method of clause 56 or 57, wherein X and Y are bonds to Z, Z comprises
X' is a bond to Z", Y' is a bond to Z', and Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain.
67. The method of clause 66, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
68. The method of any one of clauses 56 to 67, wherein L' is of the formula (X)
wherein each R13 and R13' is independently selected from the group consisting of H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 7-membered heteroaryl, -OR14,-OC(O)R14, -NR14R14', -NR14C(O)R15, -C(O)R14, -C(O)OR14, and -C(O)NR14R14', wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl is independently optionally substituted by halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR16; each R14, R14', R15, and R16 is independently selected from the group consisting of H, D, hydroxyl, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl; and h is an integer from 1 to 10; or the formula XI or XII
wherein j is an integer from 0 to 30.
69. The method of clause 68, wherein each R13 and R13' is hydrogen.
70. The method of clause 68 or 69, wherein L' is present, h is 5, m is 0, n is 1, and p is 1.
71. The method of clause 68, wherein L' is of the formula XI or XII
wherein j is 7.
72. The method of any one of clauses 56 to 71, wherein the N-terminal protecting group is a photoprotecting group.
73. The method of any one of clauses 56 to 72, wherein the N-terminal protecting group is 2-(2-nitrophenyl)propyloxycarbonyl.
74. The method of any one of clauses 23 to 54 and 56 to 73, wherein at least one of a label-free and an affinity analysis of the matured, extended core binder sequence peptides is performed.
75. The method of any one of clauses 23 to 54 and 56 to 74, wherein the first or second peptide microarray comprises at least one of glass, plastic, and carbon composite.
76. The method any one of clauses 23 to 54 and 56 to 75, wherein the functionalized peptides and the cyclic peptides on the first or the second peptide microarray comprise the same number of amino acids.
77. The method of any one of clauses 23 to 54 and 56 to 76, wherein the functionalized peptides and the cyclic peptides on the first or the second peptide microarray do not include the amino acid cysteine or methionine, or histidine-proline-glutamine motifs, or amino acid repeats of 2 or more amino acids.
78. The method of any one of clauses 23 to 54 and 56 to 77, wherein the population of matured, extended core binder sequence peptides includes at least one of an N-terminal wobble synthesis oligopeptide and a C-terminal wobble synthesis oligopeptide.
79. The method of any one of clauses 23 to 54 and 56 to 78 wherein the first or second peptide microarray comprises one or more linear peptides and wherein the method further comprises the step of contacting the one or more linear peptides on the first or second peptide microarray with a protease capable of digesting the one or more linear peptides.
80. The method of clause 79 wherein the protease is an amino protease or a mixture of amino proteases.
81. The method of clause 79 wherein the protease is dipeptidyl peptidase IV, aminopeptidase m, or a combination thereof.
82. An mRNA-displayed cow antibody scaffold polypeptide comprising a peptide of interest.
83. The mRNA-displayed cow antibody scaffold polypeptide of clause 82 comprising the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCG (SEQ ID NO: 1) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
84. The mRNA-displayed cow antibody scaffold polypeptide of clause 82 comprising the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDK (SEQ ID NO: 2) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
85. The mRNA-displayed cow antibody scaffold polypeptide of clause 82 comprising the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO: 3) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
86. The mRNA-displayed cow antibody scaffold polypeptide of any one of clauses 83 to 85 wherein X* comprises a natural amino acid.
87. The mRNA-displayed cow antibody scaffold polypeptide of any one of clauses 83 to 85 wherein X* comprises a non-natural amino acid.
88. The mRNA-displayed cow antibody scaffold polypeptide of any one of clauses 83 to 87 wherein c is an integer from 1 to 40.
89. The mRNA-displayed cow antibody scaffold polypeptide of any one of clauses 83 to 87 wherein c is an integer from 1 to 10.
90. The mRNA-displayed cow antibody scaffold polypeptide of any one of clauses 83 to 89 wherein (X*)c comprises the sequence of the peptide of interest.
91. The mRNA-displayed cow antibody scaffold polypeptide of clause 90 wherein the peptide of interest is a therapeutic peptide.
92. A peptide microarray comprising a cow antibody scaffold polypeptide comprising a peptide of interest.
93. The peptide microarray of clause 92 wherein the cow antibody scaffold polypeptide comprises the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCG (SEQ ID NO: 1) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
94. The peptide microarray of clause 92 wherein the cow antibody scaffold polypeptide comprises the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDK (SEQ ID NO: 2) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
95. The peptide microarray of clause 92 wherein the cow antibody scaffold polypeptide comprises the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO: 3) wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
96. The peptide microarray of any one of clauses 93 to 95 wherein X* comprises a natural amino acid.
97. The peptide microarray of any one of clauses 93 to 95 wherein X* comprises a non-natural amino acid.
98. The peptide microarray of any one of clauses 93 to 97 wherein c is an integer from 1 to 40.
99. The peptide microarray of any one of clauses 93 to 97 wherein c is an integer from 1 to 10.
100. The peptide microarray of any one of clauses 93 to 99 wherein (X*)c comprises the sequence of the peptide of interest.
101. The peptide microarray of clause 100 wherein the peptide of interest is a therapeutic peptide.
102. The method, mRNA-displayed cow antibody scaffold polypeptide, or peptide microarray of any one of clauses 1 to 101 wherein the cow antibody scaffold polypeptide comprises a random amino acid sequence cassette and the random amino acid sequence cassette comprises the sequence of the peptide of interest.
103. The method, mRNA-displayed cow antibody scaffold polypeptide, or peptide microarray of clause 102 wherein the random amino acid sequence cassette does not include the sequence of an antibody, a fragment of an antibody, or a peptide fragment derived from an antibody.
104. The method of clause 43 or 44 wherein the butelase 1 recognition sequence is NHV.
105. The method of clause 45, 46, 66, or 67 wherein the glutamine side chain is part of the sequence [WY][DE][DE][YW]ALQ[GST]YD (SEQ ID NO:4) and the lysine side chain is part of the sequence RSKLG (SEQ ID NO:5).
106. The method of any one of clauses 1 to 22 further comprising the step of synthesizing the cow antibody scaffold polypeptide comprising the peptide of interest on a peptide microarray to mature and/or extend the peptide of interest or the step of synthesizing the peptide of interest on a peptide microarray to mature and/or extend the peptide of interest.
107. The method, mRNA-displayed cow antibody scaffold polypeptide, or peptide microarray of any one of clauses 1 to 101 wherein the cow antibody scaffold polypeptide comprises a random amino acid sequence cassette wherein the random amino acid sequence cassette comprises the sequence of the peptide of interest and wherein the random amino acid sequence cassette is used for selection of peptides of interest.
108. A cow antibody scaffold polypeptide comprising a random amino acid sequence cassette.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic drawing of a cow antibody scaffold polypeptide. Figure 1 discloses SEQ ID NOS 400 and 401, respectively, in order of appearance.
Figure 2 shows a denaturing gel of a single thrombin-binding peptide grafted on a cow antibody scaffold polypeptide.
Figure 3 shows agarose gel electrophoresis of various PCR cycles to determine detection levels of amplification products.
Figure 4 shows a Biacore sensorgram of a single cycle kinetics measurement of crude peptides from a translation mixture and derived binding constants.
Figure 5 shows Biacore sensorgrams of multiple cycle kinetics measurements of selected thrombin binders. Figure 5 discloses SEQ ID NOS 367-368, respectively, in order of appearance.
Figure 6 shows a schematic drawing of an N (N-terminal base) motif, an A (ascending beta-strand) motif, an R (random) motif, a D (descending beta-strand) motif, and a C (C-terminal base) motif (SEQ ID NO: 402) in a cow antibody scaffold polypeptide.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
wherein each R1, R2, R3 and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and a N-terminal protecting group;
R7 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R8 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R9 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain, and a non-natural amino acid side chain;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
each L' and L" is independently an optional bivalent linking group or a bond;
b is an integer from 0 to 50;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 6;
r is 0 or 1;
s is an integer from 0 to 100;
t is 0 or 1;
u is 0 or 1;
v is an integer from 0 to 100;
w is 0 or 1; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface; and *** is a point of connection to the rest of the functionalized peptide;
the method comprising the step of reacting a functionalized peptide of formula II under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L', L", *, and *** are as defined for formula I;
R10 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R11 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R12 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q' is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z';
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
g is an integer from 0 to 50; and
y is 0 or 1;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
wherein each R1, R2, R3, and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently a natural amino acid side chain or a non-natural amino acid side chain selected such that
can form a beta-sheet;
each R7 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and an N-terminal protecting group;
R8 is selected from the group consisting of hydrogen, an N-terminal capping group, and a protecting group;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
L' is an optional bivalent linking group or a bond;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 50;
r is an integer from 0 to 50;
s is an integer from 0 to 50;
t is an integer from 0 to 50;
u is an integer from 0 to 50; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface;
the method comprising the step of reacting a functionalized peptide of formula IX under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, R7, and R8, m, n, p, q, r, s, t, u, L' and * are as defined for formula VIII;
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z'; and
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
mRNA Display Using Cow Antibody Scaffold Polypeptides
Peptide Microarrays and Their Use
wherein each R1, R2, R3 and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently hydrogen or an N-terminal capping group;
each R7 is independently -OH or a C-terminal capping group;
Q is selected from the group consisting of a carbonyl, a natural amino acid side chain, and a non-natural amino acid side chain;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
L' and L" are each independently an optional bivalent linking group or a bond;
m is an integer from 0 to 6;
n is an integer from 0 to 6;
p is an integer from 0 to 100;
q is 0 or 1;
r is 0 or 1;
t is an integer from 0 to 100;
u is 0 or 1; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface;
the method comprising the step of reacting a functionalized peptide of formula II under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, Q, m, n, p, q, r, t, u, and * are as defined for formula I;
each R7 is independently selected from the group consisting of -OH, a C-terminal capping group, and
each R8 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R9 is independently -OH or a C-terminal capping group;
each X' is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
each Y' is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z';
Z' and Z" are each independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
b is an integer from 0 to 50;
and *** is a point of connection to the rest of the functionalized peptide;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;109. A method of selecting a peptide of interest, the method comprising the steps of
110. The method of clause 109 wherein the step of preparing the peptide library comprises the step of in vitro transcription of a DNA library to form an mRNA library.
111. The method of clause 109 or 110 further comprising the step of digesting the DNA library with DNase.
112. The method of clause 109 or 110 further comprising the step of conjugating the mRNA from the mRNA library to a puromycin oligonucleotide linker.
113. The method of any one of clauses 110 to 112 further comprising the step of translating in vitro the mRNA from the mRNA library to form mRNA-antibody scaffold polypeptide fusion conjugates comprising the antibody scaffold polypeptide wherein the antibody scaffold polypeptide comprises a purification tag.
114. The method of clause 113 further comprising the step of purifying the mRNA-antibody scaffold polypeptide fusion conjugates using the purification tag.
115. The method of clause 113 or 114 further comprising the step of reverse transcribing the mRNA from the mRNA library to form mRNA-DNA duplexes of the mRNA- antibody scaffold polypeptide fusion conjugates.
116. The method of clause 115 wherein the step of selecting comprises contacting the mRNA-DNA duplexes of the mRNA-antibody scaffold polypeptide fusion conjugates with the target molecule.
117. The method of clause 116 further comprising the step of regenerating the DNA from the mRNA-DNA duplexes of the mRNA-antibody scaffold polypeptide fusion conjugates.
118. The method of any one of clauses 110 to 117 wherein the DNA library is a cDNA library.
119. The method of any one of clauses 110 to 118 wherein the in vitro transcription is performed using T7 RNA polymerase.
120. The method of any one of clauses 112 to 119 wherein the puromycin oligonucleotide linker is linked to the mRNA at the 3' end of the mRNA.
121. The method of any one of clauses 113 to 120 wherein the purification tag is a FLAG tag.
122. The method of any one of clauses 110 to 121 wherein members of the DNA library comprise an RNA polymerase promoter sequence, an enhancer sequence, and a purification tag sequence.
123. The method of any one of clauses 113 to 122 wherein the purification tag is a C-terminal tag.
124. The method of any one of clauses 109 to 123 further comprising the steps of:
wherein each R1, R2, R3 and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and a N-terminal protecting group;
R7 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R8 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R9 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain, and a non-natural amino acid side chain;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
each L' and L" is independently an optional bivalent linking group or a bond;
b is an integer from 0 to 50;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 6;
r is 0 or 1;
s is an integer from 0 to 100;
t is 0 or 1;
u is 0 or 1;
v is an integer from 0 to 100;
w is 0 or 1; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface; and *** is a point of connection to the rest of the functionalized peptide;
the method comprising the step of reacting a functionalized peptide of formula II under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L', L", *, and *** are as defined for formula I;
R10 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R11 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R12 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q' is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z';
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
g is an integer from 0 to 50; and
y is 0 or 1;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
125. The method of clause 124, wherein Z comprises a moiety selected from the group consisting of an amide bond,
and
wherein d is an integer from 0 to 6, e is an integer from 0 to 6, and f is an integer from 0 to 6, and ** is a point of connection to the rest of the cyclic peptide.
126. The method of clause 124 or 125, wherein Z comprises a peptide bond, Z" comprises an N-terminal protecting group, t is 0, u is 0, and y is 0.
127. The method of clause 126, further comprising removing Z" from the rest of the functionalized peptide to cause the peptide bond to form.
128. The method of clause 124 or 125, wherein Q and X are bonds to Z, Z comprises **-S-S-**, X' is a bond to Z", Q' is a bond to Z', Z' and Z" comprise cysteine side chains, t is 1, u is 0, v is 0, w is 1, and y is 0.
129. The method of clause 128, further comprising subjecting the functionalized peptide to oxidative conditions to cause **-S-S-** to form.
130. The method of clause 124 or 125, wherein X and Y are bonds to Z, Z comprises **-S-S-**, X' is a bond to Z", Y' is a bond to Z', Z' and Z" comprise cysteine side chains, t is 1, u is 1, and y is 1.
131. The method of clause 130, further comprising subjecting the functionalized peptide to oxidative conditions to cause **-S-S-** to form.
132. The method of clause 124 or 125, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an azide, d is 1, u is 0, v is 0, w is 1, and y is 0.
133. The method of clause 132, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
134. The method of clause 124 or 125, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, d is 1, u is 1, and y is 1.
135. The method of clause 134, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
136. The method of clause 124 or 125, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an azide, e is 1, u is 0, v is 0, w is 1, and y is 0.
137. The method of clause 136, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
138. The method of clause 124 or 125, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, e is 1, u is 1, and y is 1.
139. The method of clause 138, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
140. The method of clause 124 or 125, wherein Q is a bond to Z, Z comprises
Q' is a bond to Z', Z' comprises
Z" comprises an amine, f is 1, u is 0, v is 0, w is 1, and y is 0.
141. The method of clause 140, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
142. The method of clause 124 or 125, wherein Y is a bond to Z, Z comprises
Z" comprises an amine, Y' is a bond to Z', Z' comprises
f is 1, u is 1, and y is 1.
143. The method of clause 142, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
144. The method of clause 124 or 125, wherein R4, R10, and R11 are defined such that the functionalized peptide comprises a butelase 1 recognition sequence, Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' is an asparagine or aspartic acid side chain, u is 1, and y is 1.
145. The method of clause 144, further comprising contacting the functionalized peptide with butelase 1 to cause
to form.
146. The method of clause 124 or 125, wherein Q and X are bonds to Z, Z comprises
Q' is a bond to Z', X' is a bond to Z", Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain, t is 1, u is 0, v is 0, w is 1, and y is 0.
147. The method of clause 146, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
148. The method of clause 124 or 125, wherein X and Y are bonds to Z, Z comprises
X' is a bond to Z", Y' is a bond to Z', Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain, t is 1, u is 1, and y is 1.
149. The method of clause 148, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
150. The method of any one of clauses 124 to 149, wherein each L' and L" is independently of the formula V
wherein each R13 and R13' is independently selected from the group consisting of H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 7-membered heteroaryl, -OR14, OC(O)R14, -NR14R14', -NR14C(O)R15, -C(O)R14, -C(O)OR14, and -C(O)NR14R14', wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl is independently optionally substituted by halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR16; each R14, R14', R15, and R16 is independently selected from the group consisting of H, D, hydroxyl, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl; and h is an integer from 1 to 10; or the formula VI or VII
wherein j is an integer from 0 to 30.
151. The method of clause 150, wherein each R13 and R13' is hydrogen.
152. The method of clause 150 or 151, wherein L' is present, h is 5, m is 0, n is 1, and p is 1.
153. The method of clause 150, wherein at least one of L' and L" is of the formula VI or VII
wherein j is 7.
154. The method of any one of clauses 124 to 153, wherein the N-terminal protecting group is a photoprotecting group.
155. The method of any one of clauses 124 to 154, wherein the N-terminal protecting group is 2-(2-nitrophenyl)propyloxycarbonyl.
156. The method of any one of clauses 109 to 123 further comprising the step of synthesizing the antibody scaffold polypeptide on one or more peptide microarrays wherein the antibody scaffold polypeptide comprises the peptide of interest.
157. The method of clause 156 comprising the steps of:
wherein each R1, R2, R3, and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently a natural amino acid side chain or a non-natural amino acid side chain selected such that
can form a beta-sheet;
each R7 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and an N-terminal protecting group;
R8 is selected from the group consisting of hydrogen, an N-terminal capping group, and a protecting group;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
L' is an optional bivalent linking group or a bond;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 50;
r is an integer from 0 to 50;
s is an integer from 0 to 50;
t is an integer from 0 to 50;
u is an integer from 0 to 50; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface;
the method comprising the step of reacting a functionalized peptide of formula IX under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, R7, and R8, m, n, p, q, r, s, t, u, L' and * are as defined for formula VIII;
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z'; and
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
158. The method of clause 157, wherein Z comprises a moiety selected from the group consisting of an amide bond,
and
wherein d is an integer from 0 to 6, e is an integer from 0 to 6, and f is an integer from 0 to 6, and ** is a point of connection to the rest of the cyclic peptide.
159. The method of clause 157 or 158, wherein X and Y are bonds to Z, Z comprises **-S-S-**, X' is a bond to Z", Y' is a bond to Z', and Z' and Z" comprise cysteine side chains.
160. The method of clause 159, further comprising subjecting the functionalized peptide to oxidative conditions to cause **-S-S-** to form.
161. The method of clause 157 or 158, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, and d is 1.
162. The method of clause 161, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
163. The method of clause 157 or 158, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an azide, and e is 1.
164. The method of clause 163, further comprising contacting the functionalized peptide with a copper catalyst to cause
to form.
165. The method of clause 157 or 158, wherein Y is a bond to Z, Z comprises
Y' is a bond to Z', Z' comprises
Z" comprises an amine, and f is 1.
166. The method of clause 165, further comprising contacting the functionalized peptide with a potassium ferricyanide to cause
to form.
167. The method of clause 157 or 158, wherein X and Y are bonds to Z, Z comprises
X' is a bond to Z", Y' is a bond to Z', and Z' is a glutamine side chain and Z" is a lysine side chain or Z' is a lysine side chain and Z" is a glutamine side chain.
168. The method of clause 167, further comprising contacting the functionalized peptide with a microbial transglutaminase to cause
to form.
169. The method of any one of clauses 157 to 158, wherein L' is of the formula (X)
wherein each R13 and R13' is independently selected from the group consisting of H, D, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 7-membered heteroaryl, -OR14, -OC(O)R14, -NR14R14', -NR14C(O)R15, -C(O)R14, -C(O)OR14, and -C(O)NR14R14', wherein each hydrogen atom in C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl is independently optionally substituted by halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR16; each R14, R14', R15, and R16 is independently selected from the group consisting of H, D, hydroxyl, C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl, C3-C6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-C10 aryl and 5- to 7-membered heteroaryl; and h is an integer from 1 to 10; or the formula XI or XII
wherein j is an integer from 0 to 30.
170. The method of clause 169, wherein each R13 and R13' is hydrogen.
171. The method of clause 169 or 170, wherein L' is present, h is 5, m is 0, n is 1, and p is 1.
172. The method of clause 169, wherein L' is of the formula XI or XII
wherein j is 7.
173. The method of any one of clauses 157 to 172, wherein the N-terminal protecting group is a photoprotecting group.
174. The method of any one of clauses 157 to 173, wherein the N-terminal protecting group is 2-(2-nitrophenyl)propyloxycarbonyl.
175. The method of any one of clauses 124 to 155 and 157 to 174, wherein at least one of a label-free and an affinity analysis of the matured, extended core binder sequence peptides is performed.
176. The method of any one of clauses 124 to 155 and 157 to 175, wherein the first or second peptide microarray comprises at least one of glass, plastic, and carbon composite.
177. The method any one of clauses 124 to 155 and 157 to 176, wherein the functionalized peptides and the cyclic peptides on the first or the second peptide microarray comprise the same number of amino acids.
178. The method of any one of clauses 124 to 155 and 157 to 177, wherein the functionalized peptides and the cyclic peptides on the first or the second peptide microarray do not include the amino acid cysteine or methionine, or histidine-proline-glutamine motifs, or amino acid repeats of 2 or more amino acids.
179. The method of any one of clauses 124 to 155 and 157 to 178, wherein the population of matured, extended core binder sequence peptides includes at least one of an N-terminal wobble synthesis oligopeptide and a C-terminal wobble synthesis oligopeptide.
180. The method of any one of clauses 124 to 155 and 157 to 179 wherein the first or second peptide microarray comprises one or more linear peptides and wherein the method further comprises the step of contacting the one or more linear peptides on the first or second peptide microarray with a protease capable of digesting the one or more linear peptides.
181. The method of clause 180 wherein the protease is an amino protease or a mixture of amino proteases.
182. The method of clause 180 wherein the protease is dipeptidyl peptidase IV, aminopeptidase m, or a combination thereof.
183. An mRNA-displayed antibody scaffold polypeptide comprising a peptide of interest.
184. The mRNA-displayed antibody scaffold polypeptide of clause 183 wherein the peptide of interest is a therapeutic peptide.
185. A peptide microarray comprising an antibody scaffold polypeptide comprising a peptide of interest.
186. The peptide microarray of clause 185 wherein the peptide of interest is a therapeutic peptide.
187. The method, mRNA-displayed antibody scaffold polypeptide, or peptide microarray of any one of clauses 109 to 186 wherein the antibody scaffold polypeptide comprises a random amino acid sequence cassette and the random amino acid sequence cassette comprises the sequence of the peptide of interest.
188. The method, mRNA-displayed antibody scaffold polypeptide, or peptide microarray of clause 187 wherein the random amino acid sequence cassette does not include the sequence of an antibody, a fragment of an antibody, or a peptide fragment derived from an antibody.
189. The method of clause 144 or 145 wherein the butelase 1 recognition sequence is NHV.
190. The method of clause 146, 147, 167, or 168 wherein the glutamine side chain is part of the sequence [WY][DE][DE][YW]ALQ[GST]YD (SEQ ID NO:4)and the lysine side chain is part of the sequence RSKLG (SEQ ID NO:5).
191. The method of any one of clauses 109 to 123 further comprising the step of synthesizing the antibody scaffold polypeptide comprising the peptide of interest on a peptide microarray to mature and/or extend the peptide of interest.
192. The method, mRNA-displayed antibody scaffold polypeptide, or peptide microarray of any one of clauses 109 to 186 wherein the antibody scaffold polypeptide comprises a random amino acid sequence cassette wherein the random amino acid sequence cassette comprises the sequence of the peptide of interest and wherein the random amino acid sequence cassette is used for selection of peptides of interest.
193. An antibody scaffold polypeptide comprising a random amino acid sequence cassette.
Example 1
Microarrays
EXAMPLE 2
Design of a Cow Antibody CDR H3 Scaffold Polypeptide for mRNA display
EXAMPLE 3
Construction of a Synthetic DNA Library Coding the Combinatorial Peptide Library
EXAMPLE 4
Expression of a Thrombin-Binding Peptide and a Peptide Library
Fixed modifications: carbamidomethyl (C) |
Variable modifications: Deamidated (NQ),Oxidation (M) |
Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 98% |
Matched peptides shown in Bold Red |
EXAMPLE 5
Construction of an mRNA Display Library with Stalk-and-Knob Structures and Selection of High-Affinity Target (Thrombin) Binders
1) Sequence elements of the proposed mRNA display library
2) PCR amplification of 300-53-N10 library
PCR mix | |
5X HF buffer (NEB) | 200 ul |
100 uM 300-48-1 | 10 ul 1000 pmol |
100uM 300-22-2 | 10 ul 1000 pmol |
10 uM synthetic template 300-53-N10 | 10 ul, 100 pmol. |
10 mM dNTP | 20 ul |
Water | 745 ul |
Phusion polymerase (NEB) Hot start | 5 ul |
3) Transcription of the above-generated DNA library
T7 RiboMax (Promega) transcription in 50 ul. | |
5X T7 buffer | 10 ul |
25 mM rNTP each | 12.5 ul |
244.5 ng/ul DNA library | 22.5 ul |
T7 Enzyme mix | 5 ul |
4) Puromycin spacer ligation
10X T4 RNA ligase buffer (NEB) | 5 ul |
10 mM ATP | 1.5 ul |
2939.6 ng/ul mRNA library | 10 ul |
100 uM puromycin spacer | 20 ul |
T4 RNA ligase (NEB) | 10 ul |
H2O | 3.5 ul |
5) PURExpress deltaRF123 (NEB) translation of mRNA-spacer
100 uM reverse primer 300-22-2 | 1 ul |
10 mM dNTP | 2 ul |
Rnase free H2O | 23.5 ul |
5X RT buffer (Invitrogen) | 8 ul |
0.1 M DTT | 4 ul |
RNasin (Promega) | 0.5 ul |
Superscript III (Invitrogen) | 1 ul |
7) Pre-selection
9) M270 beads capture
10) Amplify library by Phusion PCR
5X HF buffer (NEB) | 40 ul |
10 uM forward primer 300-48-1 | 4 ul |
10 uM reverse primer 300-22-2 | 4 ul |
10 mM dNTP | 4 ul |
cDNA | 147 ul |
Phusion polymerase | 1 ul |
11) Iterative rounds of selections -
12) Results and analysis
Example 6
Selection Progress Against Thrombin and Sequences of Selected Peptides
Round 1 | Round 2 | Round 3 | ||||||
sequence | counts | percentage (%) | sequence | counts | percentage (%) | sequence | counts | percentage (%) |
(SEQ ID NO:333) SQSERATPRY | 47 | 1.5E-03 | (SEQ ID NO:343) RDPGRLIFQS | 2642 | 0.1608 | (SEQ ID NO:353) RDPGRLIFQS | 170914 | 20.512 |
(SEQ ID NO:334) RFHLFILGTS | 46 | 1.4E-03 | (SEQ ID NO:344) RDPGRVIFEI | 1632 | 0.0993 | (SEQ ID NO:354) RDPGRVIFEI | 83638 | 10.038 |
(SEQ ID NO:335) PPVLPQL VLL | 44 | 1.4E-03 | (SEQ ID NO:345) RDPGRIVFNN | 781 | 0.0475 | (SEQ ID NO:355) RDPGRIVFNN | 9534 | 1.144 |
(SEQ ID NO:336) PLIHPRRGYA | 43 | 1.3E-03 | (SEQ ID NO:346) RDPYNVLISL | 278 | 0.0170 | (SEQ ID NO:356) RDPYNVLISL | 702 | 0.084 |
(SEQ ID NO:337) HRLQIATVYT | 42 | 1.3E-03 | (SEQ ID NO:347) HRLQIATVYT | 179 | 0.0109 | (SEQ ID NO:357) RSHGQFSFTF | 258 | 0.031 |
(SEQ ID NO:338) TQPKLTIEQR | 42 | 1.3E-03 | (SEQ ID NO:348) ETRIYIRFHT | 86 | 0.0052 | (SEQ ID NO:358) SLFVEYRITY | 224 | 0.027 |
(SEQ ID NO:339) HCDTLELRPA | 40 | 1.2E-03 | (SEQ ID NOS: 349 and 371, respectively) RTKIK*NGL* | 86 | 0.0052 | (SEQ ID NO:359) QMSFRFEVRV | 211 | 0.025 |
(SEQ ID NO:340) LKQTDFRTPI | 39 | 1.2E-03 | (SEQ ID NO:350) VTSTYMFMYA | 84 | 0.0051 | (SEQ ID NO:360) SIEFGLTFSF | 195 | 0.023 |
(SEQ ID NO:341) KCCTSMVIQL | 38 | 1.2E-03 | (SEQ ID NO:351) AAVRLNSSS* | 75 | 0.0046 | (SEQ ID NO:361) VTSTYMFMYA | 175 | 0.021 |
(SEQ ID NO:342) LQRAISSYSR | 38 | 1.2E-03 | (SEQ ID NO:352) VVEFHFKWCI | 75 | 0.0046 | (SEQ ID NO:362) QFHLEF AFTL | 164 | 0.020 |
Example 7
Binding Characteristics of Selected Thrombin Binders
Primer PC1:
Primer PC2: 5'-TTAACTACCCTTTTCGAACTGCGGATGGCTCCA-3'(SEQ ID NO:366).
5X HF buffer (NEB) | 200 ul |
100 uM PC1 | 10 ul 1000 pmol |
100 uM PC2 | 10 ul 1000 pmol |
10 uM synthetic template | 10 ul 100 pmol. |
10 mM dNTP | 20 ul |
Water | 745 ul |
Phusion polymerase Hot start | 5 ul |
Solution A: | 10 µl |
Solution B: | 7.5 µl |
RNAsin Ribonuclease Inhibitor: | 1 µl |
Nuclease-free H2O: | 4.5 µl |
Template dsDNA: | 2 µl, 250 ng/ul |
SEQUENCE LISTING
<110> ROCHE DIAGNOSTICS GMBH F. HOFFMANN-LA ROCHE AG
<120> COW ANTIBODY SCAFFOLD POLYPEPTIDE METHOD AND COMPOSITION
<130> P32658-WO
<140>
<141>
<150> 62/152,004
<151> 2015-04-23
<160> 402
<170> PatentIn version 3.5
<210> 1
<211> 70
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (16)..(55)
<223> Any natural or non-natural amino acid
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> This region may encompass 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30,
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> continued from above; 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, or 1 to 40 residues, wherein some positions may be absent
<400> 1
<210> 2
<211> 80
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (16)..(55)
<223> Any natural or non-natural amino acid
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> This region may encompass 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30,
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> continued from above; 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, or 1 to 40 residues, wherein some positions may be absent
<400> 2
<210> 3
<211> 82
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (16)..(55)
<223> Any natural or non-natural amino acid
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> This region may encompass 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30,
<220>
<221> MISC_FEATURE
<222> (16)..(55)
<223> continued from above; 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, or 1 to 40 residues, wherein some positions may be absent
<400> 3
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Trp or Tyr
<220>
<221> MOD_RES
<222> (2)..(3)
<223> Asp or Glu
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Trp or Tyr
<220>
<221> MOD_RES
<222> (8)..(8)
<223> Gly, Ser or Thr
<400> 4
<210> 5
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 5
<210> 6
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 6
<210> 7
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 7
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 8
<210> 9
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 9
<210> 10
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 10
<210> 11
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 11
<210> 12
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 12
<210> 13
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 13
<210> 14
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 14
<210> 15
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 15
<210> 16
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 16
<210> 17
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 17
<210> 18
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 18
<210> 19
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 19
<210> 20
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 20
<210> 21
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 21
<210> 22
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 22
<210> 23
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 23
<210> 24
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 24
<210> 25
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 25
<210> 26
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 26
<210> 27
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 27
<210> 28
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 28
<210> 29
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<4> 29
<210> 30
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 30
<210> 31
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 31
<210> 32
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 32
<210> 33
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 33
<210> 34
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 34
<210> 35
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 35
<210> 36
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 36
<210> 37
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 37
<210> 38
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 38
<210> 39
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 39
<210> 40
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 40
<210> 41
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 41
<210> 42
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 42
<210> 43
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 43
<210> 44
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 44
<210> 45
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 45
<210> 46
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 46
<210> 47
<211> 2
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 47
Ile Phe
1
<210> 48
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 48
<210> 49
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 49
<210> 50
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 50
Leu Lys Asp Asp 20
<210> 51
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 51
<210> 52
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 52
<210> 53
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 53
<210> 54
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 54
<210> 55
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 55
<210> 56
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 56
<210> 57
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 57
<210> 58
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 58
<210> 59
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 59
<210> 60
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 60
<210> 61
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 61
<210> 62
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 62
<210> 63
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 63
<210> 64
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 64
<210> 65
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 65
<210> 66
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 66
<210> 67
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 67
<210> 68
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 68
<210> 69
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 69
<210> 70
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 70
<210> 71
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 71
<210> 72
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 72
<210> 73
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 73
<210> 74
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 74
<210> 75
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 75
<210> 76
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 76
<210> 77
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 77
<210> 78
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 78
<210> 79
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 79
<210> 80
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 80
<210> 81
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 81
<210> 82
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 82
<210> 83
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 83
<210> 84
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 84
<210> 85
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 85
<210> 86
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 86
<210> 87
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 87
<210> 88
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 88
<210> 89
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 89
<210> 90
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 90
<210> 91
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 91
<210> 92
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 92
<210> 93
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 93
<210> 94
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 94
<210> 95
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 95
<210> 96
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 96
<210> 97
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 97
<210> 98
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 98
<210> 99
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 99
<210> 100
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 100
<210> 101
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 101
<210> 102
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 102
<210> 103
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 103
<210> 104
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 104
<210> 105
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 105
<210> 106
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 106
<210> 107
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 107
<210> 108
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 108
<210> 109
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 109
<210> 110
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 110
<210> 111
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 111
<210> 112
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 112
<210> 113
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 113
<210> 114
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 114
<210> 115
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 115
<210> 116
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 116
<210> 117
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 117
<210> 118
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 118
<210> 119
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 119
<210> 120
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 120
<210> 121
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 121
<210> 122
<211> 2
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 122
<210> 123
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 123
<210> 124
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 124
<210> 125
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 125
<210> 126
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 126
<210> 127
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 127
<210> 128
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 128
<210> 129
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 129
<210> 130
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 130
<210> 131
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 131
<210> 132
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 132
<210> 133
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 133
<210> 134
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 134
<210> 135
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 135
<210> 136
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 136
<210> 137
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 137
<210> 138
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 138
<210> 139
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 139
<210> 140
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 140
<210> 141
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 141
<210> 142
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 142
<210> 143
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 143
<210> 144
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 144
<210> 145
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 145
<210> 146
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 146
<210> 147
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 147
<210> 148
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 148
<210> 149
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 149
<210> 150
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 150
<210> 151
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 151
<210> 152
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 152
<210> 153
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 153
<210> 154
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 154
<210> 155
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 155
<210> 156
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 156
<210> 157
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 157
<210> 158
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 158
<210> 159
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 159
<210> 160
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 160
<210> 161
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 161
<210> 162
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 162
<210> 163
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 163
<210> 164
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 164
<210> 165
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 165
<210> 166
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 166
<210> 167
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 167
<210> 168
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 168
<210> 169
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 169
<210> 170
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 170
<210> 171
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 171
<210> 172
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 172
<210> 173
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 173
<210> 174
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 174
<210> 175
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 175
<210> 176
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 176
<210> 177
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 177
<210> 178
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 178
<210> 179
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 179
<210> 180
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 180
<210> 181
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 181
<210> 182
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 182
<210> 183
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 183
<210> 184
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 184
<210> 185
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 185
<210> 186
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 186
<210> 187
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 187
<210> 188
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 188
<210> 189
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 189
<210> 190
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 190
<210> 191
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 191
<210> 192
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 192
<210> 193
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 193
<210> 194
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 194
<210> 195
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 195
<210> 196
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 196
<210> 197
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 197
<210> 198
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 198
<210> 199
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 199
<210> 200
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 200
<210> 201
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 201
<210> 202
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 202
<210> 203
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 203
<210> 204
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 204
<210> 205
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 205
<210> 206
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 206
<210> 207
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 207
<210> 208
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 208
<210> 209
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 209
<210> 210
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 210
<210> 211
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 211
<210> 212
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 212
<210> 213
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 213
<210> 214
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 214
<210> 215
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 215
<210> 216
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 216
<210> 217
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 217
<210> 218
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 218
<210> 219
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 219
<210> 220
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 220
<210> 221
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 221
<210> 222
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 222
<210> 223
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 223
<210> 224
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 224
<210> 225
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 225
<210> 226
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 226
<210> 227
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 227
<210> 228
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 228
<210> 229
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 229
<210> 230
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 230
<210> 231
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 231
<210> 232
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 232
<210> 233
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 233
<210> 234
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 234
<210> 235
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 235
<210> 236
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 236
<210> 237
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 237
<210> 238
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 238
<210> 239
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 239
<210> 240
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 240
<210> 241
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 241
<210> 242
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 242
<210> 243
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 243
<210> 244
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 244
<210> 245
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 245
<210> 246
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 246
<210> 247
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 247
<210> 248
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 248
<210> 249
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 249
<210> 250
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 250
<210> 251
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 251
<210> 252
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 252
<210> 253
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 253
<210> 254
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 254
<210> 255
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 255
<210> 256
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 256
<210> 257
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Tyr or Phe
<220>
<221> MOD_RES
<222> (2) .. (2)
<223> Val or Ala
<400> 257
<210> 258
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 258
<210> 259
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 259
<210> 260
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 260
<210> 261
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 261
<210> 262
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 262
<210> 263
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 263
<210> 264
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 264
<210> 265
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 265
<210> 266
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 266
<210> 267
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 267
<210> 268
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 268
<210> 269
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 269
<210> 270
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 270
<210> 271
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 271
<210> 272
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 272
<210> 273
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 273
<210> 274
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 274
<210> 275
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 275
<210> 276
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 276
<210> 277
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 277
<210> 278
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 278
<210> 279
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Leu or Ser
<400> 279
<210> 280
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Lys or Arg
<220>
<221> MOD_RES
<222> (2) .. (2)
<223> Ser or Thr
<400> 280
<210> 281
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 281
<210> 282
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 282
<210> 283
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 283
<210> 284
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 284
<210> 285
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 285
<210> 286
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 286
<210> 287
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 287
<210> 288
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 288
<210> 289
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 289
<210> 290
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 290
<210> 291
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 291
<210> 292
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 292
<210> 293
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 293
<210> 294
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 294
<210> 295
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 295
<210> 296
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 296
<210> 297
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 297
<210> 298
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 298
<210> 299
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 299
<210> 300
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 300
<210> 301
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 301
<210> 302
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 302
<210> 303
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 303
<210> 304
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 304
<210> 305
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 305
<210> 306
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 306
<210> 307
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 307
<210> 308
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Phe or Tyr
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Phe, Tyr or Thr
<400> 308
<210> 309
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Tyr or Phe
<400> 309
<210> 310
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (2) .. (2)
<223> Tyr, Leu or Ser
<400> 310
<210> 311
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 311
<210> 312
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (2) .. (2)
<223> Thr or Ser
<400> 312
<210> 313
<211> 52
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (16)..(25)
<223> Any of the 20 standard amino acids
<400> 313
<210> 314
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 314
<210> 315
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 315
<210> 316
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 316
<210> 317
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 317
tgcattatca aaaagagccg cgatccgggc cgctgc 36
<210> 318
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 318
<210> 319
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (14)..(23)
<223> Any of the 20 standard amino acids
<400> 319
<210> 320
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 320
<210> 321
<211> 52
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<221> MOD_RES
<222> (16)..(25)
<223> Any of the 20 standard amino acids
<400> 321
<210> 322
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 322
<210> 323
<211> 185
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<220>
<221> modified_base
<222> (75)..(76)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (78)..(79)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (81)..(82)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (84)..(85)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (87)..(88)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (90)..(91)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (93)..(94)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (96)..(97)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (99)..(100)
<223> a, c, u, g, unknown or other
<220>
<221> modified_base
<222> (102)..(103)
<223> a, c, u, g, unknown or other
<400> 323
<210> 324
<211> 3
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 324
aug 3
<210> 325
<211> 182
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<220>
<221> modified_base
<222> (92)..(93)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (95)..(96)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (98)..(99)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (101)..(102)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (104)..(105)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (107)..(108)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (110)..(111)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (113)..(114)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (116)..(117)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (119)..(120)
<223> a, c, t, g, unknown or other
<400> 325
<210> 326
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 326
atgacgacga taagaaaaaa 20
<210> 327
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 327
taatacgact cactataggg 20
<210> 328
<211> 162
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<220>
<221> modified_base
<222> (62)..(63)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (65)..(66)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (68)..(69)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (71)..(72)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (74)..(75)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (77)..(78)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (80)..(81)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (83)..(84)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (86)..(87)
<223> a, c, t, g, unknown or other
<220>
<221> modified_base
<222> (89)..(90)
<223> a, c, t, g, unknown or other
<400> 328
<210> 329
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 329
taatacgact cactataggg ttaactttag taaggaggac agctaaatg 49
<210> 330
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 330
ttttttctta tcgtcgtcat ctttgtagtc 30
<210> 331
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 331
<210> 332
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 332
<210> 333
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 333
<210> 334
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 334
<210> 335
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 335
<210> 336
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 336
<210> 337
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 337
<210> 338
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 338
<210> 339
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 339
<210> 340
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 340
<210> 341
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 341
<210> 342
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 342
<210> 343
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 343
<210> 344
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 344
<210> 345
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 345
<210> 346
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 346
<210> 347
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 347
<210> 348
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 348
<210> 349
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 349
<210> 350
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 350
<210> 351
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 351
<210> 352
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 352
<210> 353
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 353
<210> 354
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 354
<210> 355
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 355
<210> 356
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 356
<210> 357
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 357
<210> 358
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 358
<210> 359
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 359
<210> 360
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 360
<210> 361
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 361
<210> 362
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 362
<210> 363
<211> 52
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<400> 363
<210> 364
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<400> 364
<210> 365
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic primer
<400> 365
taatacgact cactataggg ttaactttag taaggaggac agctaaatgg gttgcaccag 60
<210> 366
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic primer
<400> 366
ttaactaccc ttttcgaact gcggatggct cca 33
<210> 367
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<220>
<223> N-term Biotin
<400> 367
<210> 368
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<223> N-term Biotin
<400> 368
<210> 369
<211> 66
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polypeptide
<400> 369
<210> 370
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 370
<210> 371
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 371
<210> 372
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 372
<210> 373
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 373
<210> 374
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 374
<210> 375
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 375
<210> 376
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 376
<210> 377
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 377
<210> 378
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 378
<210> 379
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 379
<210> 380
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 380
<210> 381
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 381
<210> 382
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 382
<210> 383
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 383
<210> 384
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 384
<210> 385
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 385
<210> 386
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 386
<210> 387
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 387
<210> 388
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 388
<210> 389
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 389
<210> 390
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 390
<210> 391
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 391
<210> 392
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 392
<210> 393
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 393
<210> 394
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 394
<210> 395
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 395
<210> 396
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 396
<210> 397
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 397
<210> 398
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (5)..(5)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 398
<210> 399
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<220>
<221> MOD_RES
<222> (6)..(6)
<223> Any of the 20 standard amino acids
<220>
<223> See specification as filed for detailed description of substitutions and preferred embodiments
<400> 399
<210> 400
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 400
<210> 401
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 401
<210> 402
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic peptide
<400> 402
a) preparing a peptide library using an mRNA-displayed cow antibody scaffold polypeptide comprising the peptide of interest to be identified;
b) selecting the peptide of interest from the peptide library by contacting a target molecule with the peptide of interest wherein the target molecule is immobilized on a solid support or is in solution; and
c) identifying the amino acid sequence of the peptide of interest,
wherein the cow antibody scaffold polypeptide comprises the sequence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO: 3)
wherein (X*)c is a random sequence of amino acids, and wherein X* is an amino acid sequence and c is the number of amino acids in the random sequence of amino acids.
i) synthesizing the cow antibody scaffold polypeptide comprising the peptide of interest on a peptide microarray to at least one of mature and extend the peptide of interest; and
ii) synthesizing the peptide of interest on a peptide microarray to mature and/or extend the peptide of interest.
a) synthesizing the cow antibody scaffold polypeptide comprising the peptide of interest, or derivatives of the peptide of interest, on a first peptide microarray, wherein the derivatives of the peptide of interest include at least one alteration in the sequence of the peptide of interest selected from a single amino acid substitution, a double amino acid substitution, a deletion of one or more amino acids, and an insertion of one or more amino acids, whereby functionalized peptides are generated on the first peptide microarray;
b) forming from the functionalized peptides, wherein the functionalized peptides are in linear form, cyclic peptides of formula VIII
wherein each R1, R2, R3, and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently a natural amino acid side chain or a non-natural amino acid side chain selected such that
can form a beta-sheet;
each R7 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and an N-terminal protecting group;
R8 is selected from the group consisting of hydrogen, an N-terminal capping group, and a protecting group;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
L' is an optional bivalent linking group or a bond;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 50;
r is an integer from 0 to 50;
s is an integer from 0 to 50;
t is an integer from 0 to 50;
u is an integer from 0 to 50; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface;
the method comprising the step of reacting a functionalized peptide of formula IX under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, R7, and R8, m, n, p, q, r, s, t, u, L' and * are as defined for formula VIII;
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z'; and
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
c) exposing the cyclic peptides to the target molecule, whereby the target molecule binds to at least one cyclic peptide;
d) identifying one or more of the cyclic peptides demonstrating strong binding to the target molecule, whereby a matured core binder sequence is determined;
e) performing at least one of N-terminal and C-terminal extension of the matured core binder sequence determined in step d to provide a matured, extended core binder sequence on a second peptide microarray;
f) exposing the target molecule to the second peptide microarray comprising a population of matured, extended core binder sequence peptides generated in step e wherein the population of matured, extended core binder sequence peptides comprises cyclic peptides formed as in step b; and
g) identifying a matured, extended cyclic peptide with strong binding to the target molecule.
a) synthesizing the peptide of interest, or derivatives thereof, on a first peptide microarray, wherein the derivatives of the peptide of interest include at least one alteration in the sequence of the peptide of interest selected from a single amino acid substitution, a double amino acid substitution, a deletion of one or more amino acids, and an insertion of one or more amino acids, whereby functionalized peptides are generated on the first peptide microarray;
b) forming from the functionalized peptides, wherein the functionalized peptides are in linear form, cyclic peptides of formula I
wherein each R1, R2, R3 and R4 is independently a natural amino acid side chain or a non-natural amino acid side chain;
each R5 and R6 is independently selected from the group consisting of hydrogen, an N-terminal capping group, and a N-terminal protecting group;
R7 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R8 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R9 is selected from the group consisting of-OH, a C-terminal capping group, and a C-terminal protecting group;
Q is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain, and a non-natural amino acid side chain;
each X and Y is independently selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z, and a non-natural amino acid side chain covalently attached to Z;
Z is a group comprising a moiety selected from the group consisting of an amide bond, a disulfide bond, an isopeptide bond, a 1,2,3-triazole, and an optionally substituted 1,2-quinone;
each L' and L" is independently an optional bivalent linking group or a bond;
b is an integer from 0 to 50;
m is an integer from 0 to 6;
n is 0 or 1;
p is 0 or 1;
q is an integer from 0 to 6;
r is 0 or 1;
s is an integer from 0 to 100;
t is 0 or 1;
u is 0 or 1;
v is an integer from 0 to 100;
w is 0 or 1; and * is a point of connection connecting the cyclic peptide to an array support having a reactive surface; and *** is a point of connection to the rest of the functionalized peptide;
the method comprising the step of reacting a functionalized peptide of formula II under conditions that cause Z to form
wherein R1, R2 R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L', L", *, and *** are as defined for formula I;
R10 is selected from the group consisting of -OH, a C-terminal capping group, a C-terminal protecting group, and
each R11 is independently a natural amino acid side chain or a non-natural amino acid side chain;
R12 is selected from the group consisting of -OH, a C-terminal capping group, and a C-terminal protecting group;
Q' is selected from the group consisting of a bond, a carbonyl, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
X' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z", and a non-natural amino acid side chain covalently attached to Z";
Y' is selected from the group consisting of a bond, a natural amino acid side chain covalently attached to Z', and a non-natural amino acid side chain covalently attached to Z';
each Z' and Z" is independently selected from the group consisting of a bond, -OH, hydrogen, a thiol, an amine, a carboxylic acid, an amide, an alkyne, an azide, an optionally substituted aminophenol, a natural amino acid side chain, a non-natural amino acid side chain, an N-terminal protecting group, and a C-terminal protecting group, provided that Z' and Z" are complementary groups that combine to form Z;
g is an integer from 0 to 50; and
y is 0 or 1;
wherein the functionalized peptides and the cyclic peptides are immobilized to the reactive surface;
c) exposing the cyclic peptides to the target molecule, whereby the target molecule binds to at least one cyclic peptide;
d) identifying one or more of the cyclic peptides demonstrating strong binding to the target molecule, whereby a matured core binder sequence is determined;
e) performing at least one of N-terminal and C-terminal extension of the matured core binder sequence determined in step d to provide a matured, extended core binder sequence on a second peptide microarray;
f) exposing the target molecule to the second peptide microarray comprising a population of matured, extended core binder sequence peptides generated in step e wherein the population of matured, extended core binder sequence peptides comprises cyclic peptides formed as in step b; and
g) identifying a matured, extended cyclic peptide with strong binding to the target molecule.
a) Herstellen einer Peptidbibliothek unter Verwendung eines mRNA-Display-Kuh-Antikörper-Gerüst-Polypeptids, umfassend das zu identifizierende Peptid von Interesse;
b) Auswählen des Peptids von Interesse aus der Peptidbibliothek durch Inkontaktbringen eines Zielmoleküls mit dem Peptid von Interesse, wobei das Zielmolekül auf einem festen Träger immobilisiert oder in Lösung ist; und
c) Identifizieren der Aminosäuresequenz des Peptids von Interesse,
wobei das Kuh-Antikörper-Gerüst-Polypeptid die Sequenz MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO:3) umfasst,
wobei (X*)c eine Zufallssequenz von Aminosäuren ist, und wobei X* eine Aminosäuresequenz ist und c die Anzahl von Aminosäuren in der Zufallssequenz von Aminosäuren ist.
i) Synthetisieren des Kuh-Antikörper-Gerüst-Polypeptids, umfassend das Peptid von Interesse, auf einem Peptid-Mikroarray, um das Peptid von Interesse mindestens zur Reife zu bringen und/oder zu verlängern; und
ii) Synthetisieren des Peptids von Interesse auf einem Peptid-Mikroarray, um das Peptid von Interesse zur Reife zu bringen und/oder zu verlängern.
a) Synthetisieren des Kuh-Antikörper-Gerüst-Polypeptids, umfassend das Peptid von Interesse oder Derivate des Peptids von Interesse, auf einem ersten Peptid-Mikroarray, wobei die Derivate des Peptids von Interesse mindestens eine Veränderung in der Sequenz des Peptids von Interesse umfassen, ausgewählt aus einer einzelnen Aminosäuresubstitution, einer doppelten Aminosäuresubstitution, einer Deletion einer oder mehrerer Aminosäuren und einer Insertion einer oder mehrerer Aminosäuren, wodurch funktionalisierte Peptide auf dem erstem Peptid-Mikroarray erzeugt werden;
b) Bilden, aus den funktionalisierten Peptiden, wobei die funktionalisierten Peptide in linearer Form vorliegen, von cyclischen Peptiden der Formel VIII
wobei jeder R1, R2, R3 und R4 unabhängig voneinander eine natürliche Aminosäureseitenkette oder eine nicht natürliche Aminosäureseitenkette ist;
jeder R5 und R6 unabhängig voneinander eine natürliche Aminosäureseitenkette oder eine nicht natürliche Aminosäureseitenkette ist, so ausgewählt, dass
und
ein Beta-Faltblatt bilden können;
jeder R7 unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus Wasserstoff, einer N-terminalen Capping-Gruppe und einer N-terminalen Schutzgruppe;
R8 ausgewählt ist aus der Gruppe, bestehend aus Wasserstoff, einer N-terminalen Capping-Gruppe und einer Schutzgruppe;
jeder X und Y unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z gebunden ist;
Z eine Gruppe ist, die eine Einheit umfasst, die ausgewählt ist aus der Gruppe, bestehend aus einer Amidbindung, einer Disulfidbindung, einer Isopeptidbindung, einem 1,2,3-Triazol und einem gegebenenfalls substituierten 1,2-Chinon;
L' eine optionale zweiwertige Kupplungsgruppe oder eine Bindung ist;
m eine ganze Zahl von 0 bis 6 ist;
n 0 oder 1 ist;
p 0 oder 1 ist;
q eine ganze Zahl von 0 bis 50 ist;
r eine ganze Zahl von 0 bis 50 ist;
s eine ganze Zahl von 0 bis 50 ist;
t eine ganze Zahl von 0 bis 50 ist;
u eine ganze Zahl von 0 bis 50 ist; und * ein Verbindungspunkt ist, der das cyclische Peptid an einen Arrayträger mit einer reaktiven Oberfläche bindet;
wobei das Verfahren den Schritt des Umsetzens eines funktionalisierten Peptids der Formel IX unter Bedingungen umfasst, die -Z dazu bringen,
zu bilden,
wobei R1, R2, R3, R4, R5, R6, R7 und R8, m, n, p, q, r, s, t, u, L' und * wie für Formel VIII definiert sind;
X' ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist;
Y' ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z' gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z' gebunden ist; und
jeder Z' und Z" unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, -OH, Wasserstoff, einem Thiol, einem Amin, einer Carbonsäure, einem Amid, einem Alkin, einem Azid, einem gegebenenfalls substituierten Aminophenol, einer natürlichen Aminosäureseitenkette, einer nicht natürlichen Aminosäureseitenkette, einer N-terminalen Schutzgruppe und einer C-terminalen Schutzgruppe, mit der Maßgabe, dass Z' und Z" komplementäre Gruppen sind, die sich unter Bildung von Z vereinigen;
wobei die funktionalisierten Peptide und die cyclischen Peptide an der reaktiven Oberfläche immobilisiert sind;
c) Exponieren der cyclischen Peptide dem Zielmolekül, wobei das Zielmolekül an mindestens ein cyclisches Peptid bindet;
d) Identifizieren eines oder mehrerer der cyclischen Peptide, die eine starke Bindung an das Zielmolekül demonstrieren, wobei eine gereifte Kernbindersequenz bestimmt wird;
e) Durchführen mindestens eines von N-terminaler und C-terminaler Verlängerung der in Schritt d bestimmten gereiften Kernbindersequenz unter Bereitstellung einer gereiften, verlängerten Kernbindersequenz auf einem zweiten Peptid-Mikroarray;
f) Exponieren des Zielmoleküls dem zweiten Peptid-Mikroarray, umfassend eine Population von in Schritt e erzeugten gereiften, verlängerten Kernbindersequenzpeptiden, wobei die Population gereifter, verlängerter Kernbindersequenzpeptide cyclische Peptide, wie in Schritt b gebildet, umfasst; und
g) Identifizieren eines gereiften, verlängerten cyclischen Peptids mit starker Bindung an das Zielmolekül.
a) Synthetisieren des Peptids von Interesse oder Derivaten davon auf einem ersten Peptid-Mikroarray, wobei die Derivate des Peptids von Interesse mindestens eine Veränderung in der Sequenz des Peptids von Interesse, ausgewählt aus einer einzelnen Aminosäuresubstitution, einer doppelten Aminosäuresubstitution, einer Deletion einer oder mehrerer Aminosäuren und einer Insertion einer oder mehrerer Aminosäuren, umfassen, wobei funktionalisierte Peptide auf dem ersten Peptid-Mikroarray erzeugt werden;
b) Bilden, aus den funktionalisierten Peptiden, wobei die funktionalisierten Peptide in linearer Form vorliegen, von cyclischen Peptiden der Formel I
wobei jeder R1, R2, R3 und R4 unabhängig voneinander eine natürliche Aminosäureseitenkette oder eine nicht natürliche Aminosäureseitenkette ist;
jeder R5 und R6 unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus Wasserstoff, einer N-terminalen Capping-Gruppe und einer N-terminalen Schutzgruppe;
R7 ausgewählt ist aus der Gruppe, bestehend aus -OH, einer C-terminalen Capping-Gruppe, einer C-terminalen Schutzgruppe und
jeder R8 unabhängig voneinander eine natürliche Aminosäureseitenkette oder eine nicht natürliche Aminosäureseitenkette ist;
R9 ausgewählt ist aus der Gruppe, bestehend aus -OH, einer C-terminalen Capping-Gruppe und einer C-terminalen Schutzgruppe;
Q ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einem Carbonyl, einer natürlichen Aminosäureseitenkette und einer nicht natürlichen Aminosäureseitenkette;
jeder X und Y unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z gebunden ist;
Z eine Gruppe ist, die eine Einheit umfasst, die ausgewählt ist aus der Gruppe, bestehend aus einer Amidbindung, einer Disulfidbindung, einer Isopeptidbindung, einem 1,2,3-Triazol und einem gegebenenfalls substituierten 1,2-Chinon;
jeder L' und L" unabhängig voneinander eine optionale zweiwertige Kupplungsgruppe oder eine Bindung ist;
b eine ganze Zahl von 0 bis 50 ist;
m eine ganze Zahl von 0 bis 6 ist;
n 0 oder 1 ist;
p 0 oder 1 ist;
q eine ganze Zahl von 0 bis 6 ist;
r 0 oder 1 ist;
s eine ganze Zahl von 0 bis 100 ist;
t 0 oder 1 ist;
u 0 oder 1 ist;
v eine ganze Zahl von 0 bis 100 ist;
w 0 oder 1 ist; und * ein Verbindungspunkt ist, der das cyclische Peptid an einen Arrayträger mit einer reaktiven Oberfläche bindet; und *** ein Verbindungspunkt zum Rest des funktionalisierten Peptids ist;
wobei das Verfahren den Schritt der Umsetzung eines funktionalisierten Peptids der Formel II unter Bedingungen umfasst, die Z dazu bringen,
zu bilden
wobei R1, R2, R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L', L", * und *** wie für Formel I definiert sind;
R10 ausgewählt ist aus der Gruppe, bestehend aus -OH, einer C-terminalen Capping-Gruppe, einer C-terminalen Schutzgruppe und
jeder R11 unabhängig voneinander eine natürliche Aminosäureseitenkette oder eine nicht natürliche Aminosäureseitenkette ist;
R12 ausgewählt ist aus der Gruppe, bestehend aus -OH, einer C-terminalen Capping-Gruppe und einer C-terminalen Schutzgruppe;
Q' ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einem Carbonyl, einer natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist;
X' ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z" gebunden ist;
Y' ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, einer natürlichen Aminosäureseitenkette, die kovalent an Z' gebunden ist, und einer nicht natürlichen Aminosäureseitenkette, die kovalent an Z' gebunden ist;
jeder Z' und Z" unabhängig voneinander ausgewählt ist aus der Gruppe, bestehend aus einer Bindung, -OH, Wasserstoff, einem Thiol, einem Amin, einer Carbonsäure, einem Amid, einem Alkin, einem Azid, einem gegebenenfalls substituierten Aminophenol, einer natürlichen Aminosäureseitenkette, einer nicht natürlichen Aminosäureseitenkette, einer N-terminalen Schutzgruppe und einer C-terminalen Schutzgruppe, mit der Maßgabe, dass Z' und Z" komplementäre Gruppen sind, die sich unter Bildung von Z vereinigen;
g eine ganze Zahl von 0 bis 50 ist und
y 0 oder 1 ist;
wobei die funktionalisierten Peptide und die cyclischen Peptide an der reaktiven Oberfläche immobilisiert sind;
c) Exponieren der cyclischen Peptide dem Zielmolekül, wobei das Zielmolekül an mindestens ein cyclisches Peptid bindet;
d) Identifizieren eines oder mehrerer der cyclischen Peptide, die eine starke Bindung an das Zielmolekül demonstrieren, wobei eine gereifte Kernbindersequenz bestimmt wird;
e) Durchführen mindestens eines von N-terminaler und C-terminaler Verlängerung der in Schritt d bestimmten gereiften Kernbindersequenz unter Bereitstellung einer gereiften, verlängerten Kernbindersequenz auf einem zweiten Peptid-Mikroarray;
f) Exponieren des Zielmoleküls dem zweiten Peptid-Mikroarray, umfassend eine Population von in Schritt e erzeugten gereiften, verlängerten Kernbindersequenzpeptiden, wobei die Population gereifter, verlängerter Kernbindersequenzpeptide cyclische Peptide, wie in Schritt b gebildet, umfasst; und
g) Identifizieren eines gereiften, verlängerten cyclischen Peptids mit starker Bindung an das Zielmolekül.
a) préparation d'une banque de peptides à l'aide d'un polypeptide d'échafaudage d'anticorps bovin produit par mRNA-display comprenant le peptide d'intérêt à identifier ;
b) sélection du peptide d'intérêt à partir de la banque de peptides par la mise en contact d'une molécule cible avec le peptide d'intérêt où la molécule cible est immobilisée sur un support solide ou est en solution ; et
c) identification de la séquence d'acides aminés du peptide d'intérêt,
dans lequel le polypeptide d'échafaudage d'anticorps bovin comprend la séquence MGCTSVHQETKKYQS(X*)cSYTYNYEHVDVWGCGSADYKDDDDKKK (SEQ ID NO : 3)
où (X*)c est une séquence aléatoire d'acides aminés, et où X* est une séquence d'acides aminés et c est le nombre d'acides aminés dans la séquence aléatoire d'acides aminés.
i) la synthèse du polypeptide d'échafaudage d'anticorps bovin comprenant le peptide d'intérêt sur un microréseau peptidique pour au moins une parmi la maturation et l'extension du peptide d'intérêt ; et
ii) la synthèse du peptide d'intérêt sur un microréseau peptidique pour maturer et/ou étendre le peptide d'intérêt.
a) synthèse du polypeptide d'échafaudage d'anticorps bovin comprenant le peptide d'intérêt, ou de dérivés du peptide d'intérêt, sur un premier microréseau peptidique, dans lequel les dérivés du peptide d'intérêt comprennent au moins une altération dans la séquence du peptide d'intérêt sélectionnée parmi une seule substitution d'acide aminé, une double substitution d'acides aminés, une délétion d'un ou de plusieurs acides aminés et une insertion d'un ou de plusieurs acides aminés, où les peptides fonctionnalisés sont générés sur le premier microréseau peptidique ;
b) formation à partir des peptides fonctionnalisés, où les peptides fonctionnalisés sont sous forme linéaire, des peptides cycliques de formule VIII
dans laquelle chaque R1, R2, R3 et R4 est indépendamment une chaîne latérale d'acides aminés naturelle ou une chaîne latérale d'acides aminés non naturelle ;
chaque R5 et R6 est indépendamment une chaîne latérale d'acides aminés naturelle ou une chaîne latérale d'acides aminés non naturelle sélectionnée de telle sorte que
puissent former un feuillet bêta ;
chaque R7 est sélectionné indépendamment dans le groupe constitué par l'hydrogène, un groupe de coiffage N-terminal et un groupe de protection N-terminal ;
R8 est sélectionné dans le groupe constitué par l'hydrogène, un groupe de coiffage N-terminal et un groupe de protection ;
chaque X et Y est sélectionné indépendamment dans le groupe constitué par une liaison, une chaîne latérale d'acides aminés naturelle attachée de façon covalente à Z, et une chaîne latérale d'acides aminés non naturelle attachée de façon covalente à Z ;
Z est un groupe comprenant un fragment sélectionné dans le groupe constitué par une liaison amide, une liaison disulfure, une liaison isopeptidique, un 1,2,3-triazole et une 1,2-quinone optionnellement substituée ;
L' est un groupe de liaison bivalente optionnel ou une liaison ;
m est un entier de 0 à 6 ;
n représente 0 ou 1 ;
p représente 0 ou 1 ;
q est un entier de 0 à 50 ;
r est un entier de 0 à 50 ;
s est un entier de 0 à 50 ;
t est un entier de 0 à 50 ;
u est un entier de 0 à 50 ; et * est un point de connexion connectant le peptide cyclique à un support de réseau ayant une surface réactive ;
le procédé comprenant l'étape de réaction d'un peptide fonctionnalisé de formule IX dans des conditions qui provoquent Z à former
dans laquelle R1, R2, R3, R4, R5, R6, R7 et R8, m, n, p, q, r, s, t, u, L' et * sont tels que définis pour la formule VIII ;
X' est sélectionné dans le groupe constitué par une liaison, une chaîne latérale d'acides aminés naturelle attachée de façon covalente à Z" et une chaîne latérale d'acides aminés non naturelle attachée de façon covalente à Z" ;
Y' est sélectionné dans le groupe constitué par une liaison, une chaîne latérale d'acides aminés naturelle attachée de manière covalente à Z' et une chaîne latérale d'acides aminés non naturelle attachée de manière covalente à Z' ; et
chaque Z' et Z" est sélectionné indépendamment dans le groupe constitué par une liaison, -OH, un hydrogène, un thiol, une amine, un acide carboxylique, un amide, un alcyne, un azide, un aminophénol optionnellement substitué, une chaîne latérale d'acides aminés naturelle, une chaîne latérale d'acides aminés non naturelle, un groupe protecteur N-terminal et un groupe protecteur C-terminal, à condition que Z' et Z" soient des groupes complémentaires qui se combinent pour former Z ;
où les peptides fonctionnalisés et les peptides cycliques sont immobilisés à la surface réactive ;
c) exposition des peptides cycliques à la molécule cible, de sorte que
la molécule cible se lie à au moins un peptide cyclique ;
d) identification d'un ou plusieurs des peptides cycliques démontrant une liaison forte à la molécule cible, où une séquence de liant de noyau mature est déterminée ;
e) réalisation d'au moins une extension N-terminale et C-terminale de la séquence de liant de noyau mature déterminée à l'étape d afin de fournir une séquence de liant de noyau étendue et mature sur un second microréseau peptidique ;
f) exposition de la molécule cible au second microréseau peptidique comprenant une population de peptides d'une séquence de liant de noyau étendus et matures générés à l'étape e où la population de peptides de séquence de liant de noyau étendus et matures comprend des peptides cycliques formés comme à l'étape b ; et
g) identification d'un peptide cyclique étendu et mature, avec une forte liaison à la molécule cible.
a) synthèse du peptide d'intérêt, ou de dérivés de celui-ci, sur un premier microréseau peptidique, où les dérivés du peptide d'intérêt comprennent au moins une altération dans la séquence du peptide d'intérêt sélectionnée parmi une seule substitution d'acide aminé, une double substitution d'acide aminé, une délétion d'un ou plusieurs acides aminés et une insertion d'un ou plusieurs acides aminés, où les peptides fonctionnalisés sont générés sur le premier microréseau peptidique ;
b) formation à partir des peptides fonctionnalisés, où les peptides fonctionnalisés sont sous forme linéaire, de peptides cycliques de formule I
dans laquelle chaque R1, R2, R3 et R4 est indépendamment une chaîne latérale d'acides aminés naturelle ou une chaîne latérale d'acides aminés non naturelle ;
chaque R5 et R6 est indépendamment sélectionné dans le groupe constitué par l'hydrogène, un groupe de coiffage N-terminal et un groupe de protection N-terminal ;
R7 est sélectionné dans le groupe constitué par -OH, un groupe de coiffage C-terminal, un groupe de protection C-terminal, et
chaque R8 est indépendamment une chaîne latérale d'acides aminés naturelle ou une chaîne latérale d'acides aminés non naturelle ;
R9 est sélectionné dans le groupe constitué par -OH, un groupe de coiffage C-terminal et un groupe de protection C-terminal ;
Q est sélectionné dans le groupe constitué par une liaison, un carbonyle, une chaîne latérale d'acides aminés naturelle et une chaîne latérale d'acides aminés non naturelle ;
chaque X et Y est sélectionné indépendamment dans le groupe constitué par une liaison, une chaîne latérale d'acides aminés naturelle attachée de façon covalente à Z et une chaîne latérale d'acides aminés non naturelle attachée de façon covalente à Z ;
Z est un groupe comprenant un fragment sélectionné dans le groupe constitué par une liaison amide, une liaison disulfure, une liaison isopeptidique, un 1,2,3-triazole et une liaison 1,2-quinone optionnellement substituée ;
chaque L' et L" est indépendamment un groupe de liaison bivalente optionnelle ou une liaison ;
b est un entier de 0 à 50 ;
m est un entier de 0 à 6 ;
n représente 0 ou 1 ;
p représente 0 ou 1 ;
q est un entier de 0 à 6 ;
r représente 0 ou 1 ;
s est un entier de 0 à 100 ;
t représente 0 ou 1 ;
u représente 0 ou 1 ;
v est un entier de 0 à 100 ;
w représente 0 ou 1 ; et * est un point de connexion reliant le peptide cyclique à un support de réseau ayant une surface réactive ; et *** est un point de connexion au reste du peptide fonctionnalisé ;
le procédé comprenant l'étape de réaction d'un peptide fonctionnalisé de formule II dans des conditions qui provoquent Z à
former
dans laquelle R1, R2, R3, R4, R5, R6, m, n, p, q, r, s, t, v, w, L' L", * et *** sont tels que définis pour la formule I ;
R10 est sélectionné dans le groupe constitué par -OH, un groupe de coiffage C-terminal, un groupe de protection C-terminal, et
chaque R11 est indépendamment une chaîne latérale d'acides aminés naturelle ou une chaîne latérale d'acide aminée non naturelle ;
R12 est sélectionné dans le groupe constitué par -OH, un groupe de coiffage C-terminal et un groupe de protection C-terminal ;
Q' est sélectionné dans le groupe constitué par une liaison, d'un carbonyle, une chaîne latérale d'acides aminés naturelle attachée de manière covalente à Z" et une chaîne latérale d'acides aminés non naturelle attachée de manière covalente à Z" ;
X' est sélectionné dans le groupe constitué par une liaison, une chaîne latérale d'acides aminés naturel attachée de façon covalente à Z" et une chaîne latérale d'acides aminés non naturels attachée de façon covalente à Z" ;
Y' est sélectionné dans le groupe constitué d'une par une liaison, d'une chaîne latérale d'acides aminés naturelle attachée de manière covalente à Z' et une chaîne latérale d'acides aminés non naturels attachée de manière covalente à Z' ;
chaque Z' et Z" est sélectionné indépendamment dans le groupe constitué par une liaison, -OH, un hydrogène, un thiol, une amine, un acide carboxylique, un amide, un alcyne, un azide, un aminophénol optionnellement substitué, une chaîne latérale d'acides aminés naturelle, une chaîne latérale d'acides aminés non naturelle, un groupe protecteur N-terminal et un groupe protecteur C-terminal, à condition que Z' et Z" soient des groupes complémentaires qui se combinent pour former Z ;
g est un entier de 0 à 50 ; et
y représente 0 ou 1 ;
où les peptides fonctionnalisés et les peptides cycliques sont immobilisés à la surface réactive ;
c) exposition des peptides cycliques à la molécule cible, de sorte que la molécule cible se lie à au moins un peptide cyclique ;
d) identification d'un ou plusieurs des peptides cycliques démontrant une liaison forte à la molécule cible, où une séquence de liant de noyau mature est déterminée ;
e) réalisation d'au moins une des opérations suivantes : extension N-terminale et C-terminale de la séquence de liant de noyau mature déterminée à l'étape d afin de fournir une séquence de liant de noyau étendue et mature sur un second microréseau peptidique ;
f) exposition de la molécule cible au second microréseau peptidique comprenant une population de peptides d'une séquence de liant de noyau étendus et matures générés à l'étape e où la population de peptides de séquence de liant de noyau étendus et matures comprend des peptides cycliques formés comme à l'étape b ; et
g) identification d'un peptide cyclique étendu et mature, avec une forte liaison à la molécule cible.
REFERENCES CITED IN THE DESCRIPTION
Patent documents cited in the description
Non-patent literature cited in the description