U.S. patent application number 15/645623 was filed with the patent office on 2017-10-26 for method of using a peptide library.
This patent application is currently assigned to DAIICHI SANKYO COMPANY, LIMITED. The applicant listed for this patent is Daiichi Sankyo Company, Limited. Invention is credited to Takako KIMURA, Naoya SHINOZAKI, Tohru TAKAHASHI, Takeshi TAKIZAWA.
Application Number | 20170305988 15/645623 |
Document ID | / |
Family ID | 46602822 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170305988 |
Kind Code |
A1 |
TAKAHASHI; Tohru ; et
al. |
October 26, 2017 |
METHOD OF USING A PEPTIDE LIBRARY
Abstract
The present invention provides a peptide selected from the
following (i) and (ii): (i) a peptide having the amino acid
sequence represented by SEQ ID NO: 1 in the Sequence Listing; and
(ii) a peptide having an amino acid sequence derived from the amino
acid sequence represented by SEQ ID NO: 1 in the Sequence Listing
by the conservative amino acid substitution, deletion, addition, or
insertion of 1 to 28 (inclusive) amino acids except at the 1st Xaa
to the 11th Xaa counting from the amino terminus.
Inventors: |
TAKAHASHI; Tohru; (Tokyo,
JP) ; SHINOZAKI; Naoya; (Tokyo, JP) ;
TAKIZAWA; Takeshi; (Tokyo, JP) ; KIMURA; Takako;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Daiichi Sankyo Company, Limited |
Tokyo |
|
JP |
|
|
Assignee: |
DAIICHI SANKYO COMPANY,
LIMITED
|
Family ID: |
46602822 |
Appl. No.: |
15/645623 |
Filed: |
July 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13956916 |
Aug 1, 2013 |
9708382 |
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15645623 |
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PCT/JP2012/052304 |
Feb 1, 2012 |
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13956916 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B 40/02 20130101;
C07K 14/525 20130101; C07K 14/7151 20130101; C40B 40/10
20130101 |
International
Class: |
C07K 14/525 20060101
C07K014/525; C07K 14/715 20060101 C07K014/715; C40B 40/10 20060101
C40B040/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 2, 2011 |
JP |
2011-020559 |
Claims
1. A method for identifying a peptide or peptide derivative that
binds to a target molecule, comprising the following steps: (i)
contacting peptides or peptide derivatives contained in a peptide
library with the target molecule, wherein said peptide library
comprises a plurality of peptides or peptide derivatives, wherein
each peptide or peptide derivative has the amino acid sequence
represented by SEQ ID NO: 1, wherein each amino acid at the 1st Xaa
to the 11th Xaa of SEQ ID NO: 1 counting from the amino terminus is
one of the following amino acids in approximately equal frequencies
within the library: Ala, Glu, Gln, Asp, Asn, His, Trp, Arg, Lys,
Val, Leu, Ile, Phe, Tyr, Ser, Met, Gly, and Thr, but is not
cysteine or proline, and wherein the peptide library comprises at
least one peptide selected from the group consisting of: (a) a
peptide which binds to EphA2 (ephrin type-A receptor 2) and has the
sequence of SEQ ID NO: 2, 3, or 4; (b) a peptide which binds to
EGFR-Fc (epidermal growth factor receptor--Fc receptor) and has the
sequence of SEQ ID NO: 5, 6, or 7; (c) a peptide which binds VEGF
(vascular endothelial growth factor) and has the sequence of SEQ ID
NO: 8, 9, 10, 11, 12, or 13; and (d) a peptide which binds TNF-a
(tumor necrosis factor alpha) and has the sequence of SEQ ID NO:
14, 15, or 16 ; and (ii) recovering a peptide or peptide derivative
binding to the target molecule.
2. The method of claim 1, wherein the peptide library is a phage
display library, a ribosome display library, or a nucleic acid
display library.
3. The method of claim 1, wherein the peptides or peptide
derivatives are immobilized on a carrier.
4. The method of claim 1, wherein the peptides or peptide
derivatives are expressed on the surface of a eukaryotic or
prokaryotic cell, viral particle, virus-like particle, phage, or
phagemid.
5. The method of claim 1, wherein the target molecule is an
endogenous substance presented in a human or non-human animal
individual or is an exogenous substance.
6. The method of claim 1, wherein the target molecule is an
endogenous or exogenous enzyme, receptor, receptor ligand, humoral
factor, cytokine, biopolymer, cell, pathogen, toxin, and a
substance derived therefrom.
7. The method of claim 6, wherein the substance derived therefrom
is a fragment, decomposition product, metabolite, or processed
product which can be involved in directly or indirectly in the
onset or exacerbation of a disease.
8. The method of claim 1, wherein the target molecule is a molecule
other than the endogenous ligands of TACI.
9. The method of claim 1, wherein the target molecule is a
non-natural substance.
10. The method of claim 9, wherein the non-natural substance is a
mineral, polymer, plastic, or synthetic low-molecular weight
compound.
11. The method of claim 1, wherein the target molecule is
solid-phase immobilized.
12. The method of claim 1, wherein the target molecule is isolated
and/or purified from a tissue or cell.
13. The method of claim 1, wherein the target molecule is
synthesized.
14. The method of claim 13, wherein the target molecule is a
polypeptide prepared by in vitro translation.
15. The method of claim 1, wherein the target molecule is a
polypeptide prepared by expressing the polypeptide in a host cell
comprising an expressing vector encoding same and purifying said
polypeptide from the cell.
16. The method of claim 13, wherein the polypeptide comprises a
purification moiety.
17. The method of claim 16, wherein the purification moiety is a
histidine tag.
18. The method of claim 1, wherein the contacting step comprises
bringing the target molecule and the peptides or peptide
derivatives of the peptide library into proximity such that they
may interact with one another.
19. The method of claim 1, wherein a peptide or peptide derivative
of the library binds to the target molecule if the dissociation
constant is less than 100 .mu.M.
20. The method of claim 1, wherein the method comprises solid-phase
panning or liquid-phase panning.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/956,916, filed Aug. 1, 2013, which is a continuation of
International Application No. PCT/JP2012/052304, filed on Feb. 1,
2012, entitled "PEPTIDE LIBRARY", which claims the benefit of
Japanese Patent Application Number JP 2011-020559, filed on Feb. 2,
2011. The entire contents of the aforementioned applications are
hereby incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to a peptide, a derivative of
the peptide, a nucleic acid encoding the amino acid sequence of the
peptide or the derivative of the peptide, a vector comprising the
nucleic acid, a cell harboring the vector or the nucleic acid, a
method for producing the peptide or the derivative thereof
comprising culturing the cell, a peptide library comprising the
peptide and/or the derivative thereof, a method for identifying a
peptide and/or a derivative thereof binding to a target molecule, a
method for producing a peptide or a derivative thereof that binds
to a target molecule, a method for determining whether or not a
test peptide or derivative thereof binds to a target molecule, a
nucleic acid library comprising the nucleic acid, a composition
comprising the peptide or the derivative thereof, the nucleic acid,
the vector, or the cell, a reagent comprising the peptide or the
derivative thereof, the nucleic acid, the vector, or the cell,
etc.
BACKGROUND OF THE INVENTION
[0003] TACI, a member of the TNF superfamily, is known to function
as a key regulator of B cells. TACI has two cysteine-rich domains
(hereinafter, referred to as "CRDs") in its extracellular region
and binds to two ligands (APRIL and BAFF). TACI lacking N-terminal
CRD as a result of alternative splicing is also found in nature.
Reportedly, TACI C-terminal CDR (TACI_d2) alone exhibits binding
activity against both the ligands that is equivalent to the binding
activity of the whole extracellular region (TACI_d1d2) (See
International Publication No. WO 2006/052493; Melissa A.
Starovasnik, J. Biol. Chem., vol. 280 (No. 8), pp. 7218-7227
(2005).)
[0004] However, whether or not TACI_d2 or a variant thereof
exhibits high binding activity against a molecule other than the
endogenous ligands has not yet been revealed.
SUMMARY OF THE INVENTION
Technical Problem
[0005] The present inventors have conducted diligent studies on
TACI_d2 or a variant thereof, and consequently completed the
present invention, for example, by preparing a library comprising a
peptide that exhibits high binding activity against a molecule
other than the endogenous ligands.
Solution to Problem
[0006] The present invention relates to: [0007] (1)
[0008] a peptide selected from the following (i) and (ii): [0009]
(i) a peptide having the amino acid sequence represented by SEQ ID
NO: 1 in the Sequence Listing; and [0010] (ii) a peptide having an
amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 1 in the Sequence Listing, by the
conservative amino acid substitution, deletion, addition, or
insertion of 1 to 28 (inclusive) amino acids except at the 1st Xaa
to the 11th Xaa counting from the amino terminus; [0011] (2)
[0012] the peptide according to (1), wherein each of the 1st Xaa to
the 11th Xaa counting from the amino terminus is any amino acid
other than cysteine; [0013] (3)
[0014] the peptide according to (1) or (2), wherein each of the 1st
Xaa to the 11th Xaa counting from the amino terminus is any amino
acid other than proline; [0015] (4)
[0016] the peptide according to any one of (1) to (3), wherein the
conservative amino acid substitution is within any group selected
from a hydrophobic amino acid group, a neutral hydrophilic amino
acid group, an acidic amino acid group, a basic amino acid group, a
group of amino acids influencing the direction of the main chain,
and an aromatic amino acid group; [0017] (5)
[0018] the peptide according to any one of (1) to (4), wherein the
1st Xaa counting from the amino terminus is an amino acid selected
from the group consisting of glutamine, methionine, histidine,
serine, glutamic acid, asparagine, tryptophan, isoleucine, aspartic
acid, and threonine; [0019] (6)
[0020] the peptide according to any one of (1) to (5), wherein the
2nd Xaa counting from the amino terminus is an amino acid selected
from the group consisting of tryptophan, leucine, glycine,
isoleucine, methionine, aspartic acid, asparagine, and threonine;
[0021] (7)
[0022] the peptide according to any one of (1) to (6), wherein the
3rd Xaa counting from the amino terminus is an amino acid selected
from the group consisting of arginine, leucine, alanine, histidine,
threonine, valine, glutamine, glutamic acid, and serine; [0023]
(8)
[0024] the peptide according to any one of (1) to (7), wherein the
4th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of glutamic acid, arginine, lysine,
isoleucine, glutamine, tryptophan, tyrosine, glycine, and
phenylalanine; [0025] (9)
[0026] the peptide according to any one of (1) to (8), wherein the
5th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of lysine, glutamic acid, methionine,
alanine, glutamine, glycine, threonine, histidine, and tryptophan;
[0027] (10)
[0028] the peptide according to any one of (1) to (9), wherein the
6th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of methionine, tryptophan, tyrosine,
serine, phenylalanine, glutamine, and aspartic acid; [0029]
(11)
[0030] the peptide according to any one of (1) to (10), wherein the
7th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of glutamic acid, aspartic acid, alanine,
serine, lysine, arginine, histidine, and asparagine; [0031]
(12)
[0032] the peptide according to any one of (1) to (11), wherein the
8th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of lysine, glutamic acid, alanine,
tyrosine, tryptophan, methionine, leucine, arginine, and glycine;
[0033] (13)
[0034] the peptide according to any one of (1) to (12), wherein the
9th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of asparagine, serine, tyrosine, glutamic
acid, alanine, glycine, lysine, and histidine; [0035] (14)
[0036] the peptide according to any one of (1) to (13), wherein the
10th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of aspartic acid, histidine, tryptophan,
phenylalanine, asparagine, valine, and leucine; [0037] (15)
[0038] the peptide according to any one of (1) to (14), wherein the
11th Xaa counting from the amino terminus is an amino acid selected
from the group consisting of isoleucine, tyrosine, histidine,
glutamic acid, aspartic acid, leucine, alanine, methionine,
phenylalanine, and valine; [0039] (16)
[0040] the peptide according to any one of (1) to (15), wherein the
peptide has the amino acid sequence represented by any one of SEQ
ID NOs: 2 to 17 in the Sequence Listing; [0041] (17)
[0042] a derivative of the peptide according to any one of (1) to
(16), the derivative being prepared by chemically modifying or
biologically modifying the peptide; [0043] (18)
[0044] a nucleic acid described in any one of the following (i) to
(iii): [0045] (i) a nucleic acid comprising a nucleic acid
consisting of a nucleotide sequence encoding the amino acid
sequence of the peptide according to any one of (1) to (16); [0046]
(ii) a nucleic acid comprising a nucleotide sequence encoding the
amino acid sequence of the peptide according to any one of (1) to
(16); and [0047] (iii) a nucleic acid consisting of a nucleotide
sequence encoding the amino acid sequence of the peptide according
to any one of (1) to (16); [0048] (19)
[0049] a vector comprising the nucleic acid according to any one of
(18) (i) to (iii); [0050] (20)
[0051] a cell harboring the nucleic acid according to any one of
(18)(i) to (iii) or a vector according to (19); [0052] (21)
[0053] a method for producing the peptide according to any one of
(1) to (16), the method comprising the following steps (i) and
(ii): [0054] (i) culturing the cell according to (20); and [0055]
(ii) recovering the peptide from the culture obtained in step (i);
[0056] (22)
[0057] a peptide library comprising the peptide according to any
one of (1) to (16) and/or the peptide derivative according to (17);
[0058] (23)
[0059] the library according to (22), wherein the peptide and/or
the peptide derivative are prepared by a method comprising steps
(i) and (ii) according to (21); [0060] (24)
[0061] the library according to (22) or (23), wherein in the
library, the peptide or the peptide derivative as a phenotype is
linked directly or indirectly to a nucleic acid having a genotype
corresponding to the phenotype; [0062] (25)
[0063] the library according to any one of (22) to (24), wherein
the nucleic acid is a nucleic acid according to any one of(18)(i)
to (iii); [0064] (26)
[0065] the library according to any one of (22) to (25), wherein
the library is a phage display library, a ribosome display library,
or a nucleic acid display library; [0066] (27)
[0067] a method for identifying the peptide according to any one of
(1) to (16) or a derivative of the peptide according to (17)
binding to a target molecule, comprising the following steps (i)
and (ii): [0068] (i) contacting peptides or derivatives of the
peptides contained in a library according to any one of (22) to
(26) with the target molecule; and [0069] (ii) recovering the
peptide or a peptide derivative binding to the target molecule;
[0070] (28)
[0071] a method for producing the peptide according to any one of
(1) to (16) or a derivative of the peptide according to (17), that
binds to a target molecule, comprising the following steps (i) to
(iii): [0072] (i) contacting peptides or derivatives of the
peptides contained in a library according to any one of (22) to
(26) with the target molecule; [0073] (ii) recovering the peptide
or a derivative of the peptide binding to the target molecule; and
[0074] (iii) preparing, by chemical synthesis, gene recombination,
or in vitro translation, the peptide recovered in step (ii) or the
peptide derivative recovered in step (ii); [0075] (29)
[0076] a method for determining whether or not the peptide
according to any one of (1) to (16) or the derivative of the
peptide according to (17) binds to a target molecule, comprising
the following steps (i) and (ii): [0077] (i) contacting test
peptides according to any one of (1) to (16) or test derivatives of
the peptides according to (17) with the target molecule; and [0078]
(ii) determining that the test peptide or the test derivative of
the peptide is positive for binding, when the test peptide or the
test derivative of the peptide binds to the target molecule; [0079]
(30)
[0080] a method for producing the peptide according to any one of
(1) to (16) or the derivative of the peptide according to (17) that
binds to a target molecule, the method comprising the following
steps (i) to (iii): [0081] (i) contacting test peptides according
to any one of (1) to (16) or test derivatives of the peptides
according to (17) with the target molecule; [0082] (ii) determining
that the test peptide or the test derivative of the peptide is
positive for binding, when the test peptide or the test derivative
of the peptide binds to the target molecule; and [0083] (iii) when
the test peptide or derivative of the peptide has been determined
to be positive in step (ii), preparing the peptide or the
derivative of the peptide by chemical synthesis, gene
recombination, or in vitro translation; [0084] (31)
[0085] a nucleic acid library comprising the nucleic acid according
to any one of (18)(i) to (iii); [0086] (32)
[0087] the library according to (31), wherein the nucleic acid is
present in a phagemid, a cosmid, or a plasmid, or a fragment
thereof; [0088] (33)
[0089] the nucleic acid library according to (31) or (32), wherein
the nucleic acid is present in a prokaryotic or eukaryotic cell, on
viral DNA or RNA, or in a viral particle; [0090] (34)
[0091] a composition comprising the peptide according to any one of
(1) to (16), the derivative of the peptide according to (17), the
nucleic acid according to any one of (18)(i)to(iii), the vector
according to (19), or the cell according to (20); and [0092]
(35)
[0093] a reagent comprising the peptide according to any one of (1)
to (16), the derivative of the peptide according to (17), the
nucleic acid according to any one of (18)(i) to (iii), the vector
according to (19), or the cell according to (20); etc.
Advantageous Effects of Invention
[0094] The present invention provides a peptide library useful in
screening for a peptide binding to a desired target molecule.
BRIEF DESCRIPTION OF DRAWINGS
[0095] FIG. 1(A) is a diagram showing that a TACI_d2 mutant binds
to each protein as a target molecule, wherein the TACI_d2 mutant
was obtained by panning against the protein. The target molecule
used in the panning is BSA and hEphA2 for FIGS. 1(A)(1) and
1(A)(2), respectively. FIGS. 1(A)(1) and 1(A)(2) show examples of
polyclones.
[0096] FIG. 1(B) is a diagram showing that a TACI_d2 mutant binds
to each protein as a target molecule, wherein the TACI_d2 mutant
was obtained by panning against the protein. The target molecule
used in the panning is hEGFR/Fc and hErbB2/Fc for FIGS. 1(B)(3) and
1(B)(4), respectively. FIGS. 1(B)(3) and 1(B)(4) show examples of
polyclones. FIG. 1(C) is a diagram showing that a TACI_d2 mutant
binds to each protein as a target molecule, wherein the TACI_d2
mutant was obtained by panning against the protein. The target
molecule used in the panning is hVEGF, and hTNF-.alpha. for FIGS.
1(C)(5) and 1(C)(6), respectively. FIGS. 1(C)(5) and 1(C)(6) show
examples of single clones.
[0097] FIG. 2(A) is a diagram showing the comparison of specificity
among TACI_d2 mutants #1 to #3 binding to recombinant human EphA2
(hereinafter, referred to as .alpha.-EphA2 TACI_d2 #1 to #3) and
wild-type TACI_d2. hEphA2 and BSA were used as solid-phase
immobilized proteins for FIGS. 2(A)(1) and 2(A)(2), respectively.
FIG. 2(B) is a diagram showing the comparison of specificity among
TACI_d2 mutants #1 to #3 binding to recombinant human EphA2
(hereinafter, referred to as .alpha.-EphA2 TACI_d2 #1 to #3) and
wild-type TACI_d2. hBAFF and mEphA2/Fc were used as solid-phase
immobilized proteins for FIGS. 2(B)(3) and 2(B)(4),
respectively.
[0098] FIG. 3(A) is a diagram showing the comparison of binding
activity against human EphA2-expressing cells between .alpha.-EphA2
TACI_d2 #1 and #2. FIG. 3(A)(1) and 3(A)(2) show results of
assaying .alpha.-EphA2 TACI_d2 #1 (.alpha.-EphA2 #1) and
.alpha.-EphA2 TACI_d2 #2 (.alpha.-EphA2 #2), respectively. Human
EphA2-expressing cells (a) and human ErbB2-expressing cells (b)
were each used as cells to be compared. .alpha.-EphA2 TACI_d2 #1
and #2 (.alpha.-EphA2 #1 and .alpha.-EphA2 #2) specifically bound
to human EphA2-expressing cells.
[0099] FIG. 3(B) is a diagram showing the comparison of binding
activity against human EphA2-expressing cells between .alpha.-EphA2
TACI_d2 #3 and wild-type TACI_d2. FIG. 3(B)(3) and 3(B)(4) show
results of assaying .alpha.-EphA2 TACI_d2 #3 (.alpha.-EphA2 #3) and
wild-type TACI_d2 (WT), respectively. Human EphA2-expressing cells
(a) and human ErbB2-expressing cells (b) were each used as cells to
be compared.
[0100] FIG. 4 is a list of the amino acid sequences of TACI_d2
mutants that specifically bind to target molecules. The marked
portion indicates that a corresponding amino acid Ser is absent
(deleted). WT represents a wild-type amino acid sequence. X
(-marked portion) corresponds to (the position of) Xaa in the amino
acid sequence represented by SEQ ID NO: 1.
DETAILED DESCRIPTION
[0101] The present invention provides a peptide, a derivative of
the peptide, a peptide library, a nucleic acid, a vector, a cell, a
method for producing the peptide and/or the derivative thereof, a
method for identifying a peptide and/or a derivative thereof having
desired properties, a method for producing a peptide and/or
derivative thereof having desired properties, a method for
determining whether or not a test peptide or test derivative
thereof binds to a target molecule, a nucleic acid library, a
composition, a reagent, etc. Hereinafter, various aspects of the
present invention will be described. However, the aspects of the
present invention are not limited thereto.
1. Peptide
[0102] The present invention provides a peptide.
[0103] The "peptide" of the present invention even incorporates a
"polypeptide" and a "protein" in its meaning. In the present
invention, this "peptide" even incorporates a "peptide derivative"
in its meaning.
[0104] According to one aspect of the present invention, the
peptide has the amino acid sequence represented by SEQ ID NO: 1 in
the Sequence Listing. In the amino acid sequence, each of the 1st
Xaa to the 11th Xaa counting from the amino terminus is any amino
acid, preferably any amino acid other than cysteine, more
preferably any amino acid other than cysteine and proline.
[0105] According to an even more preferred aspect of the present
invention, in the amino acid sequence of the peptide, the 1st Xaa
counting from the amino terminus (corresponding to an amino acid at
position 11 in SEQ ID NO: 1) is an amino acid selected from the
group consisting of glutamine, methionine, histidine, serine,
glutamic acid, asparagine, tryptophan, isoleucine, aspartic acid,
and threonine; the 2nd Xaa counting from the amino terminus
(corresponding to an amino acid at position 13 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of tryptophan,
leucine, glycine, isoleucine, methionine, aspartic acid,
asparagine, and threonine; the 3rd Xaa counting from the amino
terminus (corresponding to an amino acid at position 14 in SEQ ID
NO: 1) is an amino acid selected from the group consisting of
arginine, leucine, alanine, histidine, threonine, valine, glutamic
acid, and serine; the 4th Xaa counting from the amino terminus
(corresponding to an amino acid at position 15 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of glutamic acid,
arginine, lysine, isoleucine, glutamine, tryptophan, tyrosine,
glycine, and phenylalanine; the 5th Xaa counting from the amino
terminus (corresponding to an amino acid at position 16 in SEQ ID
NO: 1) is an amino acid selected from the group consisting of
lysine, glutamic acid, methionine, alanine, glutamic acid, glycine,
threonine, histidine, and tryptophan; the 6th Xaa counting from the
amino terminus (corresponding to an amino acid at position 17 in
SEQ ID NO: 1) is an amino acid selected from the group consisting
of tryptophan, tyrosine, serine, phenylalanine, glutamine, and
aspartic acid; the 7th Xaa counting from the amino terminus
(corresponding to an amino acid at position 20 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of glutamic acid,
aspartic acid, alanine, serine, lysine, arginine, histidine, and
asparagine; the 8th Xaa counting from the amino terminus
(corresponding to an amino acid at position 25 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of lysine,
glutamic acid, alanine, tyrosine, tryptophan, methionine, leucine,
arginine, and glycine; the 9th Xaa counting from the amino terminus
(corresponding to an amino acid at position 28 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of asparagine,
serine, tyrosine, glutamic acid, alanine, glycine, lysine, and
histidine; the 10th Xaa counting from the amino terminus
(corresponding to an amino acid at position 31 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of aspartic acid,
histidine, tryptophan, phenylalanine, asparagine, valine, and
leucine; and the 11th Xaa counting from the amino terminus
(corresponding to an amino acid at position 32 in SEQ ID NO: 1) is
an amino acid selected from the group consisting of isoleucine,
tyrosine, histidine, glutamic acid, aspartic acid, leucine,
alanine, methionine, phenylalanine, and valine. Alternatively, each
of the 1st Xaa to the 11th Xaa counting from the amino terminus may
be an amino acid varied by conservative amino acid substitution
(which is described in detail in the other part of the present
invention) from an amino acid selected from each group described in
this paragraph.
[0106] According to an aspect of the present invention, the peptide
has an amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 1 in the Sequence Listing by the
substitution, deletion, addition, or insertion of amino acid(s)
except at the 1st Xaa to the 11th Xaa counting from the amino
terminus. The number of substituted, deleted, added, or inserted
amino acid(s) in the amino acid sequence represented by SEQ ID NO:
1 in the Sequence Listing, except at the 1st Xaa to the 11th Xaa
counting from the amino terminus, can be 1 to 28 (inclusive). The
lower limit thereof is 1. The upper limit thereof is 28, 26, 24,
22, 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, or 2 . 1 is the minimum
limit thereof.
[0107] In the amino acid sequence, each of the 1st Xaa to the 11th
Xaa counting from the amino terminus is any amino acid, preferably
any amino acid other than cysteine, more preferably any amino acid
other than cysteine and proline, even more preferably an amino acid
selected from each group described above or an amino acid varied
from the amino acid by conservative amino acid substitution.
[0108] A "conservative amino acid substitution" means the
substitution of a certain amino acid by an amino acid functionally
equivalent or similar thereto. The conservative amino acid
substitution in the peptide brings about static change to the amino
acid sequence of the peptide. For example, one or more amino acids
similar in polarity to amino acid(s) in the peptide act
functionally equivalently thereto and bring about static change to
the amino acid sequence of this peptide. In general, substitution
within a certain group can be regarded as being conservative in
terms of structure and function. As is obvious to those skilled in
the art, however, the role of a particular amino acid residue may
have an implication onthe three-dimensional structure of a molecule
containing the amino acid. For example, a cysteine residue can take
an oxidized (disulfide) form having lower polarity than that of a
reduced (thiol) form. A long aliphatic moiety in an arginine side
chain can constitute structurally and functionally important
features. Also, an aromatic ring-containing side chain (tryptophan,
tyrosine, and phenylalanine) can contribute to ion-aromatic
interactions or cation-pi interactions. In this case, the
substitution of an amino acid having such a side chain by an amino
acid belonging to an acidic or nonpolar group can be structurally
and functionally conservative. Residues such as proline, glycine,
and cysteine (disulfide form) may have a direct impact on the
three-dimensional structure of the main chain and can hardly be
substituted without structural distortion.
[0109] A conservative amino acid substitution includes, as shown
below, specific substitution based on side chain similarity (L.
Lehninger, Biochemistry, 2nd edition, pp 73-75, Worth Publisher,
New York (1975)) and typical substitution. [0110] (1) Nonpolar
amino acid group: alanine (hereinafter, referred to as "Ala" or
simply as "A"), valine (hereinafter, referred to as "Val" or simply
as "V"), leucine (hereinafter, referred to as "Leu" or simply as
"L"), isoleucine (hereinafter, referred to as "Ile" or simply as
"I"), proline (hereinafter, referred to as "Pro" or simply as "P"),
phenylalanine (hereinafter, referred to as "Phe" or simply as "F"),
tryptophan (hereinafter, referred to as "Trp" or simply as "W"),
and methionine (hereinafter, referred to as "Met" or simply as "M")
[0111] (2) Uncharged polar amino acid group: glycine (hereinafter,
referred to as "Gly" or simply as "G"), serine (hereinafter,
referred to as "Ser" or simply as "S"), threonine (hereinafter,
referred to as "Thr" or simply as "T"), cysteine (hereinafter,
referred to as "Cys" or simply as "C"), tyrosine (hereinafter,
referred to as "Tyr" or simply as "Y"), asparagine (hereinafter,
referred to as "Asn" or simply as "N"), and glutamine (hereinafter,
referred to as "Gln" or simply as "Q") [0112] (3) Acidic amino acid
group: aspartic acid (hereinafter, referred to as "Asp" or simply
as "D") and glutamic acid (hereinafter, referred to as "Glu" or
simply as "E") [0113] (4) Basic amino acid group: lysine
(hereinafter, referred to as "Lys" or simply as "K"), arginine
(hereinafter, referred to as "Arg" or simply as "R"), and histidine
(hereinafter, referred to as "His" or simply as "H")
[0114] Naturally occurring amino acids can be divided into the
following groups based on the properties of their common side
chains: [0115] (1) Hydrophobic amino acid group: norleucine, Met,
Ala, Val, Leu, and Ile [0116] (2) Neutral hydrophilic amino acid
group: Cys, Ser, Thr, Asn, and Gln [0117] (3) Acidic amino acid
group: Asp and Glu [0118] (4) Basic amino acid group: His, Lys, and
Arg [0119] (5) Group of amino acids influencing the direction of
the main chain: Gly and Pro [0120] (6) Aromatic amino acid group:
Trp, Tyr, and Phe
[0121] Hereinafter, examples of the conservative substitution will
be shown. However, the conservative amino acid substitution of the
present invention is not limited thereto.
[0122] Ala may be substituted by, for example, Val, Leu, Ile, Met,
norleucine, Pro, Phe, or Trp.
[0123] Arg may be substituted by, for example, Lys or His.
[0124] Asn may be substituted by, for example, Cys, Ser, Thr, Gln,
Tyr, or Gly.
[0125] Asp may be substituted by, for example, Glu.
[0126] Cys may be substituted by, for example, Gly, Ser, Thr, Tyr,
Asn, or Gln.
[0127] Gln may be substituted by, for example, Gly, Ser, Thr, Cys,
Tyr, or Asn.
[0128] Glu may be substituted by, for example, Asp.
[0129] Gly may be substituted by, for example, Ser, Cys, Thr, Tyr,
Asn, Gln, Pro, Asp, or Glu.
[0130] His may be substituted by, for example, Lys or Arg.
[0131] Ile may be substituted by, for example, Leu, Val, Met, Pro,
Ala, Phe, Trp, or norleucine.
[0132] Leu may be substituted by, for example, norleucine, Ile,
Val, Pro, Met, Ala, Phe, Trp, or Met.
[0133] Lys may be substituted by, for example, Arg or His.
[0134] Met may be substituted by, for example, Ala, Val, Leu, Phe,
Ile, Pro, Trp, or norleucine.
[0135] Norleucine may be substituted by, for example, Met, Ala,
Val, Leu, Ile, Pro, Phe, or Trp.
[0136] Phe may be substituted by, for example, Trp, Leu, Val, Ile,
Ala, Tyr, Pro, or Met.
[0137] Pro may be substituted by, for example, Ala, Val, Leu, Ile,
Phe, Trp, Met, or Gly.
[0138] Ser may be substituted by, for example, Thr, Cys, Asn, Gln,
Gly, or Tyr.
[0139] Thr may be substituted by, for example, Val, Ser, Gly, Cys,
Tyr, Asn, or Gln.
[0140] Trp may be substituted by, for example, Tyr, Phe, Ala, Val,
Leu, Ile, Pro, or Met.
[0141] Tyr may be substituted by, for example, Gly, Cys, Asn, Gln,
Trp, Phe, Thr, or Ser.
[0142] Val may be substituted by, for example, Ile, Leu, Met, Trp,
Phe, Ala, norleucine, or Pro.
[0143] Examples of the amino acid sequence of the peptide of the
present invention having the amino acid sequence constituted of
these amino acids can include the following, though the amino acid
sequence of the peptide of the present invention is not limited
thereto:
TABLE-US-00001 SLSCRKEQGKQYWREKMDCECASKCGNHPDICAYFCEN (SEQ ID NO: 2
in the Sequence Listing: No. 1 in FIG. 4)
SLSCRKEQGKQYLLREWDCDSCASECGSHPHYCAYFCEN (SEQ ID NO: 3 in the
Sequence Listing: No. 2 in FIG. 4)
SLSCRKEQGKMYLLKEWDCASCASACGNHPHYCAYFCEN (SEQ ID NO: 4 in the
Sequence Listing: No. 3 in FIG. 4)
SLSCRKEQGKHYLLKEYDCDSCASECGYHPDYCAYFCEN (SEQ ID NO: 5 in the
Sequence Listing: No. 4 in FIG. 4)
SLSCRKEQGKSYGAIMYDCSSCASYCGEHPWHCAYFCEN (SEQ ID NO: 6 in the
Sequence Listing: No. 5 in FIG. 4)
SLSCRKEQGKEYGAIAWDCSSCASYCGAHPFECAYFCEN (SEQ ID NO: 7 in the
Sequence Listing: No. 6 in FIG. 4)
SLSCRKEQGKNYIHQQWDCASCASECGGHPNYCAYFCEN (SEQ ID NO: 8 in the
Sequence Listing: No. 7 in FIG. 4)
SLSCRKEQGKWYMTWESDCKSCASWCGSHPFDCAYFCEN (SEQ ID NO: 9 in the
Sequence Listing: No. 8 in FIG. 4)
SLSCRKEQGKMYDLYGFDCRSCASMCGKHPDLCAYFCEN (SEQ ID NO: 10 in the
Sequence Listing: No. 9 in FIG. 4)
SLSCRKEQGKMYMVWTQDCKSCASWCGAHPVACAYFCEN (SEQ ID NO: 11 in the
Sequence Listing: No. 10 in FIG. 4)
SLSCRKEQGKIYNQYGFDCKSCASWCGKHPDMCAYFCEN (SEQ ID NO: 12 in the
Sequence Listing: No. 11 in FIG. 4)
SLSCRKEQGKIYMTWHDDCHSCASLCGSHPLFCAYFCEN (SEQ ID NO: 13 in the
Sequence Listing: No. 12 in FIG. 4)
SLSCRKEQGKDYMVFGQDCHSCASWCGKHPVACAYFCEN (SEQ ID NO: 14 in the
Sequence Listing: No. 13 in FIG. 4)
SLSCRKEQGKQYMAGHFDCNSCASRYGHHPLMCAYFCEN (SEQ ID NO: 15 in the
Sequence Listing: No. 14 in FIG. 4)
SLSCRKEQDKTYIEYGFDCRSCASGCGGHPLMCAYFCEN (SEQ ID NO: 16 in the
Sequence Listing: No. 15 in FIG. 4)
SLSCRKEQGKSYTSEWFDCASCASKYGKHPLVCAYFCEN (SEQ ID NO: 17 in the
Sequence Listing: No. 16 in FIG. 4)
[0144] In the present invention, the amino acid can be L-amino
acid, D-amino acid, or a mixture thereof (DL-amino acid) but means
L-amino acid unless otherwise specified.
[0145] In the present invention, an amino acid may be any of amino
acids other than those described above (hereinafter, these amino
acids are collectively referred to as "abnormal amino acids" for
the sake of convenience). Examples of abnormal amino acids can
include selenocysteine, N-formylmethionine, pyrrolysine,
pyroglutamic acid, cystine, hydroxyproline, hydroxylysine,
thyroxine, O-phosphoserine, desmosine, .beta.-alanine, sarcosine,
ornithine, creatine, .gamma.-aminobutyric acid, opine, theanine,
tricholomic acid, kainic acid, domoic acid, and acromelic acid,
which are found in natural peptides or proteins. Examples of
non-natural amino acids can include, but not limited to:
N-terminally protected amino acids such as Ac-amino acid, Boc-amino
acid, Fmoc-amino acid, Trt-amino acid, and Z-amino acid;
C-terminally protected amino acids such as t-butyl ester, benzyl
ester, cyclohexyl ester, and fluorenyl ester of amino acids; and
other amino acids including diamine, .omega. amino acid, .beta.
amino acid, .gamma. amino acid, Tic derivatives of amino acids, and
aminophosphonic acid.
[0146] The peptide of the present invention can be prepared by a
method for producing peptides or proteins, well known to those
skilled in the art, such as chemical synthesis, gene recombination,
or in vitro translation. Also, the peptide from the library of the
present invention or the like screened for by the identification
method of the present invention can be prepared by such a
method.
[0147] Examples of the chemical synthesis method can include, but
are not limited to, a t-butoxycarbonyl (Boc) method and a
9-fluorenylmethoxycarbonyl (Fmoc) method. The Fmoc method has
advantages such as mild deprotection conditions and the convenient
excision of peptides from resins (Fmoc solid phase peptide
synthesis: a practical approach, ed. by W. C. Chan, P. D. White
Eds., Oxford University Press, New York, 2000.).
[0148] In the present invention, the "derivative of the peptide"
and the "peptide derivative" mean a chemically modified or
biologically modified form of the peptide of the present invention.
The chemical modification means the conversion of the original
peptide into a different substance through a chemical reaction,
i.e., the formation or cleavage of an atom-atom bond, in or on the
peptide of the present invention. The biological modification means
the conversion of the original peptide into a different substance
through a biological reaction, i.e., through the use of an
organism-derived protein (enzyme, cytokine, etc.), nucleic acid
(ribozyme, etc.), cell, tissue, or organ, or a non-human individual
or by the direct or indirect action thereof, in or on the peptide
of the present invention.
[0149] This "derivative" is not particularly limited as long as the
derivative is a substance different from the original peptide.
Examples thereof can include a substance containing a naturally
occurring sugar chain or an artificially developed sugar chain, a
substance containing a polymer such as polyethylene glycol (PEG), a
substance containing a synthetic compound or a natural compound, a
labeled substance, a substance containing a moiety necessary for
solid-phase immobilization, a substance containing a signal peptide
linked to the amino terminus, a substance containing a tag for use
in purification or isolation, and a substance in which a peptide as
a phenotype is linked directly or indirectly to a genotype
corresponding to the phenotype, and combinations of two or more
thereof.
[0150] The derivative of the peptide of the present invention can
be prepared by subjecting the peptide of the present invention, as
a starting material, to a method for chemically or biologically
modifying peptides or proteins that is well known to those skilled
in the art, such as chemical reaction, biochemical reaction, or
post-translational modification. The derivative of a peptide from
the library of the present invention or the like, screened for by
the identification method of the present invention can also be
prepared by such a method. Alternatively, the post-translationally
modified peptide derivative of the present invention may be
prepared by gene recombination using a cell capable of providing
desired post-translational modification. In addition, the peptide
derivative of the present invention containing a modified amino
acid can be prepared by adding the modified amino acid to an in
vitro translation system.
[0151] Examples of the PEGylation method can include, but are not
limited to, a method involving reacting peptides or proteins with
N-hydroxysuccinimide ester (NHS)--PEG.
[0152] According to a preferred aspect, the peptide of the present
invention and the derivative thereof each bind to a target
molecule.
[0153] Examples of the form that can be taken by the peptide of the
present invention and the derivative thereof can include, butare
not limited to, an isolated form (freeze-dried preparation,
solution, etc.), a form bound with an additional molecule
(solid-phase immobilized form, fusion protein, an assembly with a
foreign molecule, form bound with a target molecule, etc.), a
physical collection containing even other peptides, etc. (including
the peptide library of the present invention), a form expressed or
displayed on cell surface (on Escherichia coli or yeast cell
surface, etc.) (including the cell of the present invention), and a
form expressed or displayed on a viral particle. A form suitable
for a purpose such as use or storage can be selected freely.
2. Nucleic Acid
[0154] The present invention provides a nucleic acid.
[0155] In the present invention, the "nucleic acid" is a
mononucleotide, an oligonucleotide, or a polynucleotide and is also
referred to as a "gene". Examples of the nucleic acid of the
present invention can include, but not limited to, DNA, cDNA, RNA,
mRNA, cRNA, probes, oligonucleotides, polynucleotides, primers, and
vectors. Also, the nucleic acid of the present invention can be any
of single-stranded nucleotides, double-stranded nucleotides, and an
hybrid of 3 or more nucleotides strands and encompasses even a
single-stranded nucleotides hybrid consisting of DNA and RNA, a
double-stranded nucleotides consisting of the nucleotide and its
complementary strand, a double-stranded hybrid consisting of
single-stranded DNA and single-stranded RNA, double-stranded RNA,
single-stranded nucleotides that may have a double-stranded
structure moiety in its molecule, etc. The nucleic acid of the
present invention may further contain one or more (artificially
developed) bases or one or more mononucleotides, other than
naturally occurring bases or mononucleotides.
[0156] Preferred examples of the nucleic acid of the present
invention can include a nucleic acid comprising nucleotides
consisting of a nucleotide sequence encoding the amino acid
sequence of the peptide of the present invention, and a nucleic
acid comprising a nucleotide sequence encoding the amino acid
sequence of the peptide of the present invention. This preferred
nucleic acid may contain a nucleotide sequence other than the
nucleotide sequence encoding the amino acid sequence of the peptide
of the present invention, and/or a non-nucleotide moiety and may be
modified chemically or biologically (which is described in the
other part of the present invention). These forms are all
encompassed by the "nucleic acid".
[0157] The nucleic acid of the present invention also encompasses a
nucleic acid consisting of a nucleotide sequence encoding the amino
acid sequence of the peptide of the present invention.
[0158] In the present invention, in the case where the amino acid
sequence of the peptide of the present invention is encoded by a
portion or the whole of the nucleotide sequence of certain
nucleotides, this nucleic acid is referred to as a "nucleic acid
encoding or corresponding to the amino acid sequence of the
peptide" and this peptide is referred to as a "peptide encoded by
or corresponding to the nucleic acid".
[0159] Examples of the nucleic acid encoding the amino acid
sequence of the peptide of the present invention can include, but
are not limited to, a nucleic acid comprising a nucleic acid
consisting of a nucleotide sequence encoding the amino acid
sequence of the peptide of the present invention, a nucleic acid
comprising a nucleotide sequence encoding the amino acid sequence
of the peptide of the present invention, and a nucleic acid
consisting of a nucleotide sequence encoding the amino acid
sequence of the peptide of the present invention.
[0160] Examples of the peptide corresponding to the nucleic acid of
the present invention can include, but not limited to, a peptide
comprising a peptide consisting of an amino acid sequence encoded
by a portion or the whole of the nucleotide sequence of the nucleic
acid of the present invention, a peptide comprising an amino acid
sequence encoded by a portion or the whole of the nucleotide
sequence of the nucleic acid of the present invention, a peptide
consisting of an amino acid sequence encoded by a portion or the
whole of the nucleotide sequence of the nucleic acid of the present
invention, and a derivative of any one of these peptides.
[0161] In the present invention, the phrase "genotype corresponding
to (the) phenotype" is also used interchangeably with the "nucleic
acid encoding the amino acid sequence of the peptide". Likewise,
the phrase "phenotype corresponding to (the) genotype" is also used
interchangeably with the "peptide encoded by the nucleic acid".
[0162] When the chemically or biologically modified form of the
nucleic acid of the present invention contains the peptide of the
present invention, this modified form is encompassed in the scope
of the "derivative of the peptide" of the present invention.
[0163] More preferred examples of the nucleic acid of the present
invention can include, of the preferred nucleic acid described
above, a nucleic acid comprising a nucleic acid consisting of a
nucleotide sequence encoding the amino acid sequence of the peptide
of the present invention binding to a target molecule, a nucleic
acid comprising a nucleotide sequence encoding the amino acid
sequence of the peptide of the present invention binding to a
target molecule, and a nucleic acid consisting of a nucleotide
sequence encoding the amino acid sequence of the peptide of the
present invention binding to a target molecule.
[0164] In the present invention, one or more codons corresponding
to each amino acid can be used for designing the nucleotide
sequence encoding the amino acid sequence. Hence, a nucleotide
sequence encoding the single amino acid sequence of a certain
peptide or protein may have a plurality of variations. For the
selection of such codons, the codons can be selected appropriately
according to the codon usage of cells (host cells) to harbor a
genotype corresponding to the peptide, i.e., a nucleic acid
comprising the nucleotide sequence, or the frequency or rate of a
plurality of codons used can be adjusted appropriately. For
example, in the case of using Escherichia coli cells as host cells,
the nucleotide sequence can be designed using codons with high
frequency in use in Escherichia coli.
[0165] The nucleic acid of the present invention can be prepared by
a method for producing a nucleic acid well known to those skilled
in the art, such as chemical synthesis or gene recombination. A
nucleic acid encoding the amino acid sequence of the peptide
recovered from the library of the present invention or the
like(including screened for, enriched, and isolated) by the
identification method of the present invention can also be prepared
by such a method.
[0166] Examples of the form that can be taken by the nucleic acid
of the present invention can include, but are not limited to, an
isolated form (freeze-dried preparation, solution, etc.), a form
bound with an additional molecule (solid-phase immobilized form,
etc.), a recombinant vector comprising the nucleic acid (the vector
of the present invention), a cell harboring the nucleic acid or the
vector (the cell of the present invention), a form contained in a
virus or a viral particle (including a form contained as the vector
of the present invention), and a physical collection containing
even other nucleic acids, etc. (including the nucleic acid library
of the present invention). A form suitable for a purpose such as
use or storage can be selected freely.
3. Vector
[0167] The present invention provides a recombinant vector
(hereinafter, also simply referred to as a "vector").
[0168] The vector of the present invention is not particularly
limited as long as the vector comprises the nucleic acid of the
present invention and serves as means for transferring the nucleic
acid of the present invention to cells, microorganisms, or
individuals. Preferred examples thereof can include nucleic acid
vectors such as phagemids, cosmids, and plasmids.
[0169] The vector of the present invention may be a virus that
infects prokaryotic cells or eukaryotic cells, or a viral
vector.
[0170] In the present invention, the "phagemid" means a bacterial
plasmid containing an origin of plasmid replication as well as the
second replication origin derived from a single-stranded
bacteriophage. A cell having this phagemid can replicate the
phagemid via a single strand replication mode in coinfection with
M13 or its analogous helper bacteriophage. Specifically,
single-stranded phagemid DNA is packaged in an infective particle
coated with a bacteriophage coat protein. In this way, the phagemid
DNA can be formed as a cloned double-stranded DNA plasmid in the
infected bacterium, while the phagemid can be formed as a
bacteriophage-like particle from the culture supernatant of the
coinfected cell. In order to infect a bacterium having F-pilus with
the DNA, the bacteriophage-like particle can be injected into the
bacterium to reform the particle itself as a plasmid.
[0171] A fusion gene comprising the nucleic acid encoding the amino
acid sequence of the peptide of the present invention and a
bacteriophage coat protein gene can be inserted to the phagemid.
Bacterial cells can be infected with the resulting phagemid and
cultured to express or display the peptide on the bacterium or a
phage-like particle or to produce a fusion protein of the peptide
and the coat protein into a phage particle or the culture
supernatant of the bacterium.
[0172] For example, a fusion gene comprising the nucleic acid
encoding the amino acid sequence of the peptide of the present
invention and a bacteriophage coat protein gene gpIII is inserted
to the phagemid. Escherichia coli can be coinfected with the
resulting phagemid and M13 or its analogous helper phage to produce
a fusion protein comprising the peptide and the coat protein into
the culture supernatant of the Escherichia coli.
[0173] Instead of the phagemid, various circular or noncircular
vectors, preferably viral vectors, may be used to express or
display the peptide encoded by the nucleotide sequence of the
nucleic acid of the present invention contained in the vector, on a
cell harboring the vector or a virus-like particle or to produce
the peptide into the culture supernatant of the cell according to a
method well known to those skilled in the art.
[0174] The vector (recombinant vector) of the present invention can
be prepared by a method well known to those skilled in the art such
as gene recombination.
[0175] Examples of the form that can be taken by the vector of the
present invention can include, but are not limited to, an isolated
form (freeze-dried preparation, solution, etc.), a form bound with
an additional molecule (solid-phase immobilized form, etc.), a form
transferred to a cell (including the recombinant cell of the
present invention), and a physical collection containing even other
vectors, etc. (including a particular aspect of the nucleic acid
library of the present invention). A form suitable for a purpose
such as use or storage can be selected freely.
4. Cell
[0176] According to one aspect, the present invention provides a
recombinant cell (hereinafter, also simply referred to as a
"cell").
[0177] The cell of the present invention is a cell that contains
the nucleic acid encoding the amino acid sequence of the peptide of
the present invention and that expresses the peptide. Any of
eukaryotic cells (including established cell lines, primary
cultured cells, and subcultured cells) and prokaryotic cells can be
used as a host cell or cell of the present invention without
particular limitations.
[0178] Examples of the prokaryotic cells can include, but are not
limited to, bacterial cells such as Escherichia coli and Bacillus
subtilis cells.
[0179] Examples of the eukaryotic cells can include animal cells,
insect cells, yeast cells, and fungal cells. Examples of the animal
cells can include, but are not limited to, monkey COS cells
(Gluzman, Y., Cell (1981), vol. 23, pp 175-182; American Type
Culture Collection No. ATCC CRL-1650), mouse fibroblasts NIH3T3
(American Type Culture Collection No. ATCC CRL-1658), Chinese
hamster ovary cells (CHO cells; American Type Culture Collection
No. ATCC CCL-61), and dihydrofolate reductase-deficient lines of
CHO cells (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA
(1980), vol. 77, pp 4126-4220).
[0180] The cell of the present invention can be prepared by
transferring the nucleic acid of the present invention or the
vector of the present invention to a host cell and can be prepared
preferably by transferring the vector of the present invention to a
host cell by transfection, transformation, transduction, or the
like.
[0181] Examples of the vector suitable for the preparation of the
cell of the present invention can include, but are not limited to,
replicons derived from a species compatible with the prokaryotic
cell, i.e., plasmids, cosmids, and phagemids containing a
replication origin and one or more nucleotide sequences selected
from a regulatory sequence, transcription initiation site, start
codon and stop codon (of translation), etc. The nucleic acid or the
vector may further contain a nucleotide sequence that can confer
phenotypic character (phenotype) selectivity to the cell harboring
the vector or the nucleic acid. Such a vector or nucleic acid is
transferred to a host cell, and the obtained cell can be cultured
to express the peptide of the present invention.
[0182] A host cell suitable for the post-translational modification
of the peptide of the present invention may be used as the cell of
the present invention. The cell of the present invention can be
used in (an aspect of) the method for preparing the peptide
derivative of the present invention.
[0183] Examples of the form that can be taken by the cell of the
present invention can include, but are not limited to, an isolated
form (frozen preparation, freeze-dried preparation, solution,
etc.), a form bound with an additional molecule (solid-phase
immobilized form, etc.), a cell harboring the nucleic acid or the
vector of the present invention (which is included in the cell of
the present invention), a cell expressing or displaying the peptide
of the present invention on its surface (which is included in the
cell of the present invention), and a physical collection
containing even other cells, etc. (including a particular aspect of
the nucleic acid library and the peptide library of the present
invention). A form suitable for a purpose such as use or storage
can be selected freely.
5. Method for producing peptide
[0184] According to an alternative aspect, the present invention
also provides a method for producing the peptide of the present
invention.
[0185] The peptide of the present invention can be prepared, as
mentioned above, by a method for producing peptides or proteins
well known to those skilled in the art, such as chemical synthesis,
gene recombination, or in vitro translation. A peptide recovered
from the library of the present invention or the like (including
screened for, enriched, and isolated) by the identification method
of the present invention can also be prepared by such a method.
[0186] According to an aspect of the present invention, the method
for producing the peptide of the present invention comprises the
following steps (1-1) and (1-2): [0187] (1-1) culturing a cell (the
cell of the present invention) that contains the nucleic acid
encoding the amino acid sequence of the peptide of the present
invention and that expresses the peptide or the like; and [0188]
(1-2) recovering the peptide from the culture.
[0189] According to an alternative aspect, the method for producing
the peptide of the present invention comprises the following steps
(2-1) and (2-2): [0190] (2-1) determining the amino acid sequence
of the peptide of the present invention that binds to a target
molecule; and [0191] (2-2) preparing a peptide consisting of the
amino acid sequence by chemical synthesis or gene
recombination.
[0192] According to a further alternative aspect, the method for
producing the peptide of the present invention comprises the
following steps (3-1) and (3-2): [0193] (3-1) preparing mRNA
corresponding to the peptide of the present invention; and [0194]
(3-2) preparing the peptide by in vitro translation with the mRNA
obtained in step (3-1) as a template.
[0195] Also, each of these production methods can be combined
appropriately with the identification method of the present
invention as a preliminary step. Specifically, first, steps
included in the identification method of the present invention are
carried out, and subsequently, steps included in the production
method of the present invention can be carried out. The method for
producing the peptide of the present invention may encompass such a
method further comprising (each step of) the identification method
of the present invention.
[0196] Such a method for producing the peptide of the present
invention comprises, for example, the following steps (4-1) to
(4-3): [0197] (4-1) contacting peptides contained in the peptide
library of the present invention with a target molecule; [0198]
(4-2) recovering a peptide binding to the target molecule; and
[0199] (4-3) preparing the recovered peptide by chemical synthesis,
gene recombination, or in vitro translation.
[0200] Likewise, each of these production methods can be combined
appropriately with the determination method of the present
invention as a preliminary step. Specifically, first, steps
included in the determination method of the present invention are
carried out, and subsequently, steps included in the production
method of the present invention can be carried out. The method for
producing the peptide of the present invention or the like may
encompass such a method further comprising (each step of) the
determination method of the present invention.
[0201] Such a method for producing the peptide of the present
invention or the like comprises, for example, the following steps
(5-1) to (5-3): [0202] (5-1) contacting test peptides of the
present invention with a target molecule; [0203] (5-2) determining
that the test peptide is positive for binding when the test peptide
binds to the target molecule, and [0204] (5-3) when the test
peptide has been determined to be positive in step (5-2), preparing
the peptide by chemical synthesis, gene recombination, or in vitro
translation.
[0205] The present invention also provides a method for producing
the derivative of the peptide (peptide derivative)of the present
invention. The peptide derivative of the present invention can be
prepared by the method described above (method for producing the
peptide) and then subjecting the prepared peptide to chemical
reaction, biochemical reaction, post-translational modification, or
the like.
[0206] Examples of the method for producing the peptide derivative
of the present invention can include, but are not limited to, a
method comprising each of the steps (1-1) and (1-2), (2-1) and
(2-2), (3-1) and (3-2), (4-1) to (4-3), or (5-1) to (5-3), or the
like and may further comprise the step of preparing the peptide
derivative of the present invention using the peptide of the
present invention as a starting material (hereinafter, referred to
as a "derivative preparation step").
[0207] Alternatively, a cell capable of providing desired
post-translational modification may be used in the method for
producing the peptide of the present invention to prepare the
peptide derivative of the present invention as a peptide provided
with the desired post-translational modification. In this case, for
example, the cell capable of providing desired post-translational
modification can be used as the cell in the steps (1-1) and (1-2)
or as a cell (or host cell) applied to the gene recombination in
the steps (2-2), (4-3) and (5-3) to prepare the peptide derivative
provided with the desired post-translational modification, though
the method for preparing the post-translationally modified form of
the peptide (as an aspect of the method for producing the peptide
derivative) of the present invention is not limited thereto.
6. Library
[0208] The present invention provides a library.
[0209] In the present invention, the "library" means a physical
collection of molecules that are analogous, but not identical, to
one another. The molecules contained in this collection can
coexist, for example, in one container or may be present in a
physically isolated manner as groups or individual molecules in
different containers or at different sites on a solid-phase
support. A plurality of libraries may be contained in one
collection.
[0210] The library of the present invention is not limited by any
means as long as the library is a physical collection containing
non-identical peptides and/or nucleic acids of the present
invention. Examples thereof can include a phage display library, a
ribosome display library, and a nucleic acid display library.
[0211] The "phage display" means a technique (method and means
therefor) of linking foreign peptides or proteins to the coat
proteins of filamentous phages or the like and expressing or
displaying the resulting fusion proteins on phage-like particles.
Also, the recovery (including screening, enrichment, and isolation)
of nucleic acids corresponding to the peptides or proteins using
this technique is encompassed in the scope of the "phage display".
The phage display library is one aspect of the library of the
present invention used in this technique.
[0212] The "ribosome display" means a technique (method and means
therefor) of expressing or displaying peptides or proteins in the
form of complexes comprising three molecules (mRNA-ribosome-peptide
or protein), which are formed during the translation reaction of in
vitro translation. In this context, the peptides or proteins are
translation products of the mRNAs. Also, the recovery (including
screening, enrichment, and isolation) of nucleic acids
corresponding to the peptides or proteins using this technique is
encompassed in the scope of the "ribosome display". The ribosome
display library is an alternative aspect of the library of the
present invention used in this technique.
[0213] The "nucleic acid display" means a technique (method and
means therefor) of expressing or displaying peptides or proteins in
the form of complexes comprising a nucleic acid (synonymous with
nucleic acids) and peptides or proteins encoded by the nucleic acid
(Keefe, A.D and Szostak, J.W., Nature, vol. 410 (2001), pp
715-718). Also, the recovery (including screening, enrichment, and
isolation) of a nucleic acid encoding the amino acid sequence of
the peptides or proteins using this technique is encompassed in the
scope of the "nucleic acid display". The nucleic acid display
library is a further alternative aspect of the library of the
present invention used in this technique.
[0214] Examples of the nucleic acid display can include, but not
limited to, mRNA display (Yamaguchi, J. et al., Nucleic Acids
Research, vol. 37, No. 16 e108, pp 1-13 (2009)).
[0215] The mRNA display is a technique of displaying peptides or
proteins in the form of complexes comprising mRNAs and their
translation products peptides or proteins associated via
intervening moieties (Keefe, A.D and Szostak, J.W., Nature, vol.
410 (2001), pp 715-718).
[0216] In the present invention, a physical collection of cells a
physical collection of microorganisms (including viruses, phages,
phage-like molecules, particles of any of them, etc.), and a
physical collection of naturally occurring or artificially
developed vectors (including phagemids, cosmids, plasmids, etc.),
which comprise a physical collection containing non-identical
peptides and/or nucleic acids of the present invention, as well as
a physical collection of fragments thereof and a physical
collection of chemically and/or biologically modified forms thereof
are also encompassed in the scope of the "library".
[0217] In the present invention, as mentioned above, one or more
codons corresponding to each amino acid can be used for designing
the nucleotide sequence encoding the amino acid sequence. Hence, a
nucleotide sequence encoding the single amino acid sequence of a
certain peptide or protein may have a plurality of variations. For
the selection of such codons, the codons can be selected
appropriately according to the codon usage of cells (host cells) to
harbor a genotype corresponding to the peptide, i.e., a nucleic
acid comprising the nucleotide sequence, or the frequency or rate
of a plurality of codons used can be adjusted appropriately.
Accordingly, in the nucleic acid library of the present invention,
each nucleic acid comprising a nucleotide sequence encoding the
single amino acid sequence may have a plurality of variations.
Specifically, the nucleic acid library of the present invention may
comprise a physical collection containing nucleic acids, each
comprising a nucleotide sequence encoding the amino acid sequence
of a certain peptide. This physical collection of the nucleic acids
encoding the amino acid sequence of the particular peptide may form
in itself one nucleotides library.
[0218] The library of the present invention contains a plurality of
molecules that are analogous, but not identical, to one another.
The (number of) types of analogous molecules contained in the
library are referred to as the "diversity of (the) library". For
example, the diversity of a library consisting of 100 types of
analogous molecules is 10.sup.2. In the present invention, the
diversity of the library is not particularly limited and preferably
has a higher value.
[0219] The diversity of the peptide library for use in the
identification method of the present invention and the method for
producing the peptide, comprising the steps of the identification
method is 1.times.10.sup.5 or higher, 2.times.10.sup.5 or higher,
5.times.10.sup.5 or higher, 1.times.10.sup.6 or higher,
2.times.10.sup.6 or higher, 5.times.10.sup.6 or higher,
1.times.10.sup.7 or higher, 2.times.10.sup.7 or higher,
5.times.10.sup.7 or higher, 1.times.10.sup.8 or higher,
2.times.10.sup.8 or higher, 5.times.10.sup.8 or higher,
1.times.10.sup.9 or higher, 2.times.10.sup.9 or higher,
5.times.10.sup.9 or higher, 1.times.10.sup.10 or higher,
2.times.10.sup.10 or higher, 5.times.10.sup.10 or higher,
1.times.10.sup.11 or higher, 2.times.10.sup.11 or higher,
5.times.10.sup.11 or higher, 1.times.10.sup.12 or higher,
2.times.10.sup.12 or higher, 5.times.10.sup.12 or higher,
1.times.10.sup.13 or higher, 2.times.10.sup.13 or higher,
5.times.10.sup.13 or higher, 1.times.10.sup.14 or higher,
2.times.10.sup.14 or higher, 5.times.10.sup.14 or higher,
1.times.10.sup.15 or higher, 2.times.10.sup.15 or higher,
5.times.10.sup.15 or higher, 1.times.10.sup.16 or higher,
2.times.10.sup.16 or higher, 5.times.10.sup.16 or higher, or
1.times.10.sup.17 or higher. Such diversity of the library is not
limited to an actual measured value and may be a theoretical
value.
7. Identification Method
[0220] The present invention provides a method for identifying a
peptide and/or a peptide derivative binding to a target
molecule.
[0221] (1) Target Molecule
[0222] In the present invention, the "target molecule" means a
substance to which the peptide of the present invention binds and
also means an endogenous substance present in a human or nonhuman
animal individual or an exogenous substance incorporated in vivo
into the individual. The target molecule of the present invention
is preferably any of endogenous or exogenous enzymes, receptors,
ligands of the receptors, humoral factors (e.g., cytokines), other
biopolymers, signal transducers, cells, pathogens, toxins, and
substances derived from any one or more thereof, for example,
fragments, decomposition products, metabolites, or processed
products thereof, which can be involved in directly or indirectly
in the onset or exacerbation of a disease that may affect the
individual, or exhibits correlation or inverse correlation with the
disease. Alternatively, the target molecule of the present
invention may be any of non-natural substances such as minerals,
polymers, plastics, and synthetic low-molecular-weight
compounds.
[0223] The target molecule of the present invention is used for
screening for a peptide from the peptide library of the present
invention that binds to the target molecule. The target molecule
may be a full-length molecule or a fragment thereof, or a
derivative thereof, with any amino acid, peptide, protein, sugar
chain, polymer, carrier, or the like added thereto. Alternatively,
the target molecule may be solid-phase immobilized.
[0224] (2) Preparation of Target Molecule
[0225] The target molecule of the present invention can be isolated
and/or purified, for use, from a tissue or a cell affected with a
disease. Also, the target molecule of the present invention can be
prepared by a method for producing peptides or proteins well known
to those skilled in the art, such as chemical synthesis, gene
recombination, or in vitro translation. From the target molecule
thus obtained, the derivative as mentioned above may be prepared,
if necessary.
[0226] In the present invention, the target peptide or protein can
be prepared by, for example: in vitro translation, i.e., a method
involving incubating a nucleic acid (such as DNA or cDNA)
corresponding to this peptide or protein or a vector containing the
nucleic acid in a solution containing an enzyme, a substrate, an
energetic material, etc., necessary for transcription and
translation to synthesize the desired peptide or protein in vitro;
gene recombination, i.e., a method involving transferring the
nucleic acid or the vector to prokaryotic or eukaryotic cells (host
cells), culturing the obtained recombinant cells, and then
recovering the desired peptide or protein from the culture; or
chemical synthesis.
[0227] In the case where the target molecule is a protein present
or a domain thereof on a cell membrane, the molecule can also be
prepared as a secreted protein by expressing, in an appropriate
host-vector system, a fusion protein comprising the extracellular
region of this protein or domain linked to an immunoglobulin (Ig)
constant region.
[0228] The nucleic acid corresponding to the target molecule can be
obtained by, for example, an expression cloning method, though the
obtainment method is not limited thereto. The expression cloning
method involves constructing an expression library of cDNAs
comprising nucleotide sequences encoding the amino acid sequences
of peptides or proteins, and performing polymerase chain reaction
(hereinafter, referred to as "PCR"; Saiki, R. K., et al., Science
(1988), vol. 239, pp 487-489) with this cDNA library as a template
using primers specifically amplifying the full length or partial
length of the cDNAs to clone cDNAs corresponding to the peptides or
proteins.
[0229] Examples of kits or reagents applicable to the in vitro
translation can include Rapid Translation System (RTS) manufactured
by Roche Diagnostics K.K.
[0230] Prokaryotic or eukaryotic cells applicable as host cells for
preparing the cell of the present invention can be selected
appropriately as the host cells for gene recombination.
[0231] The recombinant cell (cell harboring the nucleic acid or the
vector) obtained by gene recombination can be cultured according to
a method well known to those skilled in the art and can be allowed
to produce the desired peptide or protein into the culture or into
the cell.
[0232] The medium for use in this culture can be selected
appropriately from among those routinely used according to the host
cells. In the case of using Escherichia coli cells as host cells,
for example, an LB medium can be supplemented, if necessary, with
an antibiotic (e.g., ampicillin) and IPTG and subjected to the
culture.
[0233] The desired peptide or protein produced intracellularly or
extracellularly from the recombinant cell by this culture can be
purified and isolated by the appropriate combination of
fractionation approaches known in the art using, for example, its
physical, chemical, and/or biological properties.
[0234] Examples of the fractionation approaches can include, but
are not limited to, salting out, treatment with a protein
precipitant, dialysis, ultrafiltration, molecular sieve (gel
filtration) chromatography, adsorption chromatography, ion-exchange
chromatography, affinity chromatography, partition chromatography,
and hydrophobic chromatography.
[0235] Alternatively, a moiety useful for purification may be
linked or added to the peptide or protein in advance. As a result,
the desired peptide or protein can be purified efficiently. For
example, a histidine tag consisting of 6 residues can be linked to
the peptide or protein in advance to efficiently purify the desired
peptide or protein by nickel affinity chromatography.
Alternatively, an IgG Fc region can be liked thereto in advance to
efficiently purify the desired peptide or protein by protein A
affinity chromatography.
[0236] (3) Contact of Peptide and/or Peptide Derivative with Target
Molecule
[0237] The identification method of the present invention comprises
the step of contacting peptides and/or derivatives thereof with the
target molecule. In this context, the peptides and/or the
derivatives thereof may be contained in the peptide library.
Specifically, the identification method of the present invention
may comprise the step of contacting peptides and/or derivatives
thereof contained in the peptide library with the target
molecule.
[0238] In the present invention, the term "contacting" means that
two or more substances are brought into proximity so that two or
more of these substances can be interacted with each other.
Examples of the interaction can include, but not limited to:
covalent bonds, coordinate bonds, metal-metal bonds, ionic bonds,
metallic bonds, hydrogen bonds, and Van der Waals bond
(hereinafter, these bonds are referred to as "chemical bonds");
interactions based on electrostatic interactions such as bonds
based on Coulomb force, interionic interactions, hydrogen bonds,
dipolar interactions, and Van der Waals force (hereinafter, these
interactions are referred to as "intermolecular force"); and other
interactions, charge-transfer interactions, transannular
interactions, hydrophobic interactions, and association of peptides
and biomolecules. In the present invention, the "two or more
substances" are not particularly limited as long as the substances
include the target molecule and a test substance. The test
substance is not particularly limited as long as the substance
binds to the target molecule. Examples thereof can include the
peptide of the present invention, the derivative of the peptide, a
solid-phase carrier with the peptide or the peptide derivative
immobilized thereon, and a cell, a viral particle, or a virus-like
particle (including phages and phagemids) expressing or displaying
the peptide or the peptide derivative. This test substance may be
expressed or displayed on the surface of a eukaryotic or
prokaryotic cell, on a viral particle or a virus-like particle, or
in a ribosome- or nucleic acid-linked form by phage display,
ribosome display, nucleic acid display, or the like. [0239] (4)
Screening
[0240] The identification method of the present invention comprises
the step of screening for a peptide and/or a peptide derivative
having desired properties, preferably a peptide and/or a peptide
derivative binding to the target molecule.
[0241] In the present invention, the terms "binding" or "bound"
means that two or more substances are in proximity or in an
associated state with each other under a certain condition to the
extent that these substances can be interacted (which is described
in the other part of the present invention) with each other.
[0242] In the present invention, test substances are contacted with
target molecules under a certain condition. Subsequently, a test
substance nonspecifically adsorbed to the target molecule and a
test substance unbound or unadsorbed to the target molecule are
removed from a test substance-containing fraction. If a test
substance is present in the resulting fraction, this test substance
can be regarded as "binding" to the target molecule.
[0243] When mere nonspecific adsorption occurs between two or more
substances, the "binding" can be regarded as not occurring between
these substances. In addition, when two or more substances
contacted with each other are neither in proximity nor in an
associated state with each other to the extent that these
substances can be interacted (which is described in the other part
of the present invention) with each other, the "binding" can be
regarded as not occurring between the substances.
[0244] For example, a fluorescence antibody method (direct or
indirect method), radioimmunoassay, enzyme immunoassay (homogeneous
or heterogeneous method), ELISA, or ELISPOT, which performs assay
by flow cytometry or the like is widely used as a method for
determining the "binding" between an antibody and an antigen. In
these methods, the presence or absence of the "binding" between the
peptide or the derivative thereof and the target molecule can be
determined in the same way as in the determination of the "binding"
between an antibody and an antigen except that the test antibody
and the antigen are replaced with the peptide of the present
invention or the derivative thereof and the target molecule.
[0245] Also, the presence or absence of the "binding" can be
determined by measuring an index for binding activity or affinity.
Examples of the index for binding affinity can include a
dissociation constant and an association constant.
[0246] Provided that the chemical equilibrium between a molecule A
and an A-binding substance B is defined as follows:
ABA+B
the dissociation constant (Kd) of chemical dissociation thereof can
be calculated according to the following expression:
Kd=[A][B]/[AB]
[0247] wherein [A], [B], and [AB] represent the concentrations of
the molecule A, the substance B, and an assembly AB, respectively;
Kd represents the ratio of the molecule A and the substance B
dissociated from each other to the undissociated assembly AB; and
the reciprocal of Kd represents an association constant (Ka).
[0248] The "dissociation constant" used in the present invention
means mainly the equilibrium dissociation constant of the peptide
and/or the peptide derivative for binding to a certain target
molecule.
[0249] In the present invention, the dissociation constant can be
calculated by measuring the concentrations of dissociated
substances (peptide, peptide derivative, target molecule, etc.) and
undissociated substances (aseembly of the peptide and/or the
peptide derivative and the target molecule, etc.). The method for
determining and calculating the dissociation constant is not
particularly limited as long as the method is well known to those
skilled in the art. Examples thereof can include a method using
surface plasmon resonance and an isothermal titration calorimetry
method.
[0250] In the method using surface plasmon resonance, the
interaction between the target molecule and the peptide and/or the
derivative thereof binding thereto can be determined and calculated
as follows: a series of association and dissociation reactions is
detected by surface plasmon resonance at a plurality of peptide
concentrations; the obtained series of association and dissociation
reactions is analyzed; and the dissociation constant is calculated
from various rate constants thus obtained.
[0251] Examples of the determination-calculation system of surface
plasmon resonance can include, but not limited to, Biacore system
(GE Healthcare Japan Corp.). Procedures for the method using
Biacore system are as follows: target molecules are immobilized
onto a sensor chip of Biacore system by amine coupling; the target
molecules are contacted with peptides at a plurality of peptide
concentrations; the interaction therebetween is detected by surface
plasmon resonance; a series of association and dissociation
reactions is drawn in a sensorgram with time as the abscissa
against the amount of binding (RU) as the ordinate; the sensorgram
drawn at the plurality of peptide concentrations is fit to a 1:1
Langmuir model using BlAevaluation software (manufactured by GE
Healthcare Japan Corp.) to calculate various rate parameters; and
the dissociation constant is calculated from various rate
parameters thus calculated.
[0252] In the isothermal titration calorimetry method, the
interaction between the target molecule and the peptide and/or the
derivative thereof binding thereto can be determined and calculated
as follows: a peptide solution is added dropwise to a target
molecule-containing solution (and vice versa); the quantity of heat
generated by the interaction is measured to draw a binding
isotherm; and a dissociation constant (K.sub.D), stoichiometry of
the reaction (N), an enthalpy change (.DELTA.H), and an entropy
change (.DELTA.S) are obtained from the binding isotherm.
[0253] Examples of the system of directly measuring a very small
thermal change (exothermic change or endothermic change) associated
with an intermolecular interaction can include, but not limited to,
MicroCal system (GE Healthcare Japan Corp.). Procedures for the
method using MicroCal system are as follows: a ligand solution is
titrated to each sample cell kept at constant temperature, and
stirred; heat generation or absorption directly proportional to the
amount of binding take places through the intermolecular
interaction to change the temperature of the solution in the sample
cell; a temperature difference (.DELTA.T) from a reference cell is
detected by a cell feedback network (CFB); the reference cell or
the sample cell is heated until .DELTA.T reaches 0; a feedback
power required to maintain .DELTA.T=0 is measured to obtain the
quantity of heat generated or absorbed through the interaction; the
amount of heat generated is plotted as an ordinate against the
molar ratio of the peptide to the target molecule as an abscissa,
and the dissociation constant is calculated from the binding
isotherm.
[0254] In the identification method and/or the determination method
(which will be described later) of the present invention, a test
substance that exhibits a dissociation constant of, for example,
100 .mu.M or smaller, 50 .mu.M or smaller, 20 .mu.M or smaller, 10
.mu.M or smaller, 5 .mu.M or smaller, 2 .mu.M or smaller, 1 .mu.M
or smaller, 500 nM (0.5 .mu.M) or smaller, 200 nM or smaller, 100
nM or smaller, 50 nM or smaller, 20 nM or smaller, 10 nM or
smaller, 5 nM or smaller, 2 nM or smaller, 1 nM or smaller, 500 pM
(0.5 nM) or smaller, 200 pM or smaller, 100 pM or smaller, 50 pM or
smaller, 20 pM or smaller, 10 pM or smaller, 5 pM or smaller, 2 pM
or smaller, or 1 pM or smaller for the target molecule can be
determined to bind to the target molecule, i.e., to be positive,
though the reference value of the dissociation constant and the
criteria for determining the presence or absence of the "binding"
are not limited thereto.
[0255] In the identification method of the present invention, the
"screening" step also serves as the step of recovering the test
substance binding to the target molecule. The product may consist
only of the test substance binding to the target molecule or may
also contain a substance that does not bind to the target molecule
as long as the test substance binding to the target molecule is
contained or enriched in the product. The target molecule-binding
test substance contained or enriched in this product may be a
single substance or may be a mixture of two or more of such
substances.
[0256] The screening step, i.e., the recovery step, in the
identification method of the present invention means the step of
recovering a fraction having the target molecule-binding substance
contained or enriched therein. This step is not particularly
limited as long as the step is performed by a
fractionation-purification method well known to those skilled in
the technical field of the present invention. The step may
comprise, for example, the steps of: separating a substance bound
with the target molecule, a substance unbound with the target
molecule and a substance nonspecifically adsorbed to the target
molecule from the (fraction containing) target molecule; or eluting
the substance bound with the target molecule (separating the
substance from the target molecule). In this step, the criteria for
determining the dissociation constant or the like do not have to be
set for the presence or absence of binding.
[0257] In the present invention, the "screening" included in the
identification method of the present invention is also referred to
as "panning". In the present invention, the "panning" means
procedures of contacting peptides and/or peptide derivatives of the
present invention with the target molecule and recovering
(including screening for, concentrating, and isolating) a peptide
and/or a peptide derivative binding to the target molecule.
[0258] A method well known to those skilled in the art can be
applied to the panning. Examples thereof can include, but not
limited to, a solid-phase panning method and a liquid-phase panning
method. The solid-phase panning method can involve, for example,
immobilizing target molecules onto a solid phase, subsequently
contacting peptides contained in a liquid phase with the target
molecules, subsequently removing a peptide unbound with the target
molecule and a nonspecifically bound peptide, and then selectively
separating a peptide bound with the target molecule from (the
target molecule immobilized on) the solid phase to screen for a
peptide having the desired binding activity, though the operation
of the solid-phase panning method is not limited thereto. The
liquid-phase panning method can involve, for example, contacting
peptides with the target molecules in a solution, subsequently
removing a peptide unbound with the target molecule and a
nonspecifically bound peptide, and then selectively separating a
peptide bound with the target molecule from the target to screen
for a peptide having the desired binding activity, though the
operation of the liquid-phase panning method is not limited
thereto.
[0259] In the identification method of the present invention, the
nucleic acid encoding the amino acid sequence of the peptide
(including even the "peptide derivative") binding to the target
molecule are efficiently screened for using a library in which a
phenotype (synonymous with a phenotypic character) is linked to a
genotype (synonymous with a genetic character) corresponding
thereto. As a result, the peptide can be prepared efficiently. The
link between the phenotype and the genotype corresponding thereto
(hereinafter, simply referred to as a "phenotype and a genotype")
may be direct or indirect.
[0260] The "direct link" between the phenotype and the genotype
means that the behaviors of the phenotype and the genotype match
with each other. Even if there is a degree of distance between the
phenotype and the genotype, for example, due to the presence of an
additional intervening moiety, this case is also encompassed in the
scope of the "direct link" as long as their behaviors match with
each other. Specifically, it is not essential that they should be
physically adjacent to each other.
[0261] In the present invention, the phrase "behaviors of the
phenotype and the genotype match with each other" means that their
behaviors match with each other in aspects such as the peptide, the
nucleic acid, the vector, the cell, the production method, the
identification method, the determination method, the peptide
library, the nucleic acid library, the composition, and the reagent
of the present invention. In these aspects, even if the "match of
the behaviors" is lost wholly or partially due to an internal
factor, an external factor, their combination, or any of other
factors over time, for the time being, until a certain point in
time, or from a certain point in time onward, this case is also
encompassed in the scope of the phrase "behaviors match with each
other".
[0262] Examples of the direct link between the phenotype and the
genotype can include a ribosome display library, a nucleic acid
display library, a peptide and/or a derivative thereof linked
directly or indirectly to the nucleic acid encoding the amino acid
sequence of the peptide of the present invention, and a peptide
library comprising this peptide and/or derivative thereof.
[0263] The "indirect link" between the phenotype and the genotype
means that a particular phenotype allows access to a genotype
corresponding to the phenotype, though their behaviors do not
always match with each other. Examples of the indirect link between
the phenotype and the genotype can include, but not limited to, a
phage display library and cDNA library for use in expression
cloning.
[0264] Even though the behavior of the peptide or the derivative
thereof as a phenotype does not match with that of the nucleic acid
as a genotype corresponding thereto in each clone contained in the
phage display library, the peptide and/or the derivative thereof
(expressed or displayed on a phage-like particle) binding to the
target molecule can be screened for through the steps, for example,
of contacting peptides and/or derivatives thereof contained in the
library with the target molecule, removing a phage-like particle
unbound with the target molecule or nonspecifically adsorbed to the
target molecule, and then selectively eluting a phage-like particle
bound with the target molecule. In addition, the corresponding
genotype, i.e., the nucleic acid encoding the amino acid sequence
of this peptide or the peptide derivative thereof can be purified
and isolated and further sequenced. The advantages of such access
from the phenotype to the genotype corresponding thereto are not
limited to the case of the "indirect link" between the phenotype
and the genotype in the phage display, etc., and can also be
relished in the case of the "direct link" therebetween.
[0265] Each step included in the identification method of the
present invention can be performed repetitively two or more times.
In particular, a new peptide library can be constructed from
peptides and/or peptide derivatives recovered in the screening
step, and then subjected to the contact and screening steps to
enrich, at a higher level, the peptide and/or the derivative
thereof binding to the target molecule. This operation can be
further repeated to enrich, at a much higher level, the peptide
and/or the derivative thereof binding to the target molecule.
Finally, the efficiency of isolation thereof can be enhanced. In
addition, this higher level of enrichment of the peptide and/or the
derivative thereof binding to the target molecule achieves
isolation of a binder having higher affinity.
[0266] Alternatively, the peptide and/or the derivative thereof
binding to the target molecule may be enriched at a higher level by
more securely separating the target-bound peptide and/or derivative
thereof from a nonspecifically bound one in the step included in
the identification method of the present invention. Examples of
such a separation method include increase in the number of the step
removing a nonspecifically bound peptide, etc., and the change of a
reagent (surfactant, etc.) for use in the removal of the
nonspecifically bound peptide to a stronger one.
[0267] The peptide of the present invention or the derivative
thereof can take any form of a monomer, a homo- or hetero-dimer, a
trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer,
and a multimer composed of 9 or more monomers.
[0268] The number of the molecule of the peptide of the present
invention or the derivative thereof binding to one target molecule
or one target site can be any of 1, 2, 3, 4, 5, 6, 7, 8, and 9 or
more. This peptide or derivative thereof can bind to the target
molecule, in any form of a monomer, a homo- or hetero-dimer, a
trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer,
and a multimer composed of 9 or more monomers.
[0269] The number of the target molecule or the target site to
which one molecule of the peptide of the present invention or the
derivative thereof binds can be any of 1, 2, 3, 4, 5, 6, 7, 8, and
9 or more.
8. Composition
[0270] The present invention provides a composition.
[0271] The composition of the present invention comprises the
peptide, the peptide derivative, the nucleic acid , the vector, or
the cell of the present invention.
[0272] The composition comprising the peptide of the present
invention or the derivative thereof (including those displayed on
the surface of the cell of the present invention) can be used for
detecting a target molecule to which the peptide or the derivative
thereof binds.
[0273] The composition comprising the nucleic acid, the vector, or
the cell of the present invention can be used for preparing a
peptide having an amino acid sequence encoded by the nucleotide
sequence of the nucleic acid, or the nucleic acid contained in the
vector, or the cell of the present invention. Also, this
composition can be used for detecting a nucleic acid, a vector, a
cell, etc., containing the nucleic acid.
[0274] The composition can comprise, if necessary, for example, a
buffer, a salt, a metal, an antiseptic, a surfactant, and a
substance for reducing or preventing damage suffered on the
peptide, the peptide derivative, the nucleic acid, the vector, or
the cell of the present invention by a preparation method such as
freezing or freeze drying.
9. Reagent
[0275] The present invention provides a reagent.
[0276] The reagent of the present invention comprises the peptide,
the peptide derivative, the nucleic acid, the vector, or the cell
of the present invention.
[0277] The reagent comprising the peptide of the present invention
or the derivative thereof can be used for detecting a target
molecule to which the peptide or the derivative thereof binds.
[0278] For example, the amount of HER2 protein present in a tumor
tissue is measured by a test called immunohistochemical study
(IHC). The test result is assessed in the range of 0 (negative) to
3+ (strongly positive). A tumor patient with an IHC score of 3+ is
likely to benefit from treatment with Herceptin, while a tumor
patient with an IHC score of 0 or 1+ may be unlikely to benefit
from this treatment. A tumor patient with an IHC score of 2+ often
undergoes an additional test called fluorescence in situ
hybridization (FISH) in order to more accurately determine whether
the tumor is HER2-positive. The FISH method measures the number of
gene copies and determines a tumor containing a large number of
HER2 gene copies to be HER2-positive.
[0279] The reagent comprising the nucleic acid, the vector, or the
cell of the present invention can be used for detecting a nucleic
acid, a vector, a cell, etc., containing the nucleic acid.
[0280] The reagent of the present invention may be a
composition.
[0281] A kit comprising the reagent is also encompassed by the
reagent of the present invention.
[0282] Also, the peptide or the peptide derivative of the present
invention, the cell displaying the peptide or the peptide
derivative, etc., can be used as an element recognizing a substance
such as a target molecule, in a biosensor for the substance.
10. Determination Method
[0283] The present invention provides even a method for determining
whether or not a test substance binds to a target molecule.
[0284] The determination method of the present invention can employ
the same steps as in the steps included in the identification
method of the present invention or steps appropriately modified
from these steps. The test substance, however, which is subjected
to this determination method, does not have to be contained in a
collection such as a library. For example, a test peptide or a test
peptide derivative subjected to the determination method is not
limited to the peptide or the peptide derivative contained in the
peptide library and may be a single peptide or peptide derivative
separated from other peptides, a mixture containing them, or the
like. Specifically, the identification method of the present
invention is suitable mainly as a method for screening for one or
more having desired properties from the physical collection of test
substances, whereas the determination method of the present
invention is also suitable as an assay method for examining whether
or not a particular test substance has desired properties.
[0285] In the determination method of the present invention, for
example, determination in the step of determining whether or not
the test substance binds to the target molecule can be performed on
the basis of whether or not the test substance satisfies conditions
regarding an index for affinity such as a dissociation constant. In
the screening step, as in the identification method of the present
invention, the test substance can be determined to be positive if
the test substance is recovered as a substance binding to the
target molecule.
EXAMPLES
[0286] Hereinafter, the present invention will be described more
specifically with reference to Examples. However, the present
invention is not limited thereto.
Example 1
[0287] Preparation of Randomly Mutated TACI_d2 Phage Library
1) Synthesis of Randomly Mutated TACI_d2 Oligonucleotide
[0288] The following oligonucleotide (183 bp) was synthesized:
[0289] 5'-G CTG CAC ACT GTA GGA GAA GAC TGG GCC CAG CCG GCC AGC CTG
AGT TGC CGT AAA GAA CAG GGC AAG NNN TAT NNN NNN NNN NNN NNN GAC TGC
NNN AGC TGC GCG AGC NNN TGT GGA NNN CAT CCT NNN NNN TGC GCG TAT TTT
TGC GAA AAC GCG GCC GCG AGT CCA CGT TCC ATC GGT CA-3' (SEQ ID NO:
18 in the Sequence Listing; "N" in the nucleotide sequence
represents any base selected from A, T, G, and C).
[0290] The sequence has a 5'-terminal primer-binding region, a
restriction enzyme Sfil recognition sequence (Sfil site), a
randomly mutated TACI_d2 coding sequence, a Notl site, and a
3'-terminal primer-binding region in this order from its 5' end.
Also, the sequences each represented by NNN contain codons encoding
18 amino acids (Ala, Glu, Gln, Asp, Asn, His, Trp, Arg, Lys, Val,
Leu, Ile, Phe, Tyr, Ser, Met, Gly, and Thr) other than Cys and Pro,
at almost equal probabilities (3.0% to 8.2% for each). As a result,
the calculational diversity of randomly mutated TACI_d2 is
6.4.times.10.sup.13.
2) Preparation of Escherichia coli TG-1 Strain Having Randomly
Mutated TACI_d2 Phagemid Vector
[0291] PCR was performed with the oligonucleotides (a total of 2
.mu.g) synthesized in the preceding paragraph 1) as a template
using the following two types of primers (synthesized by Sigma Life
Science, Sigma-Aldrich Corp.):
TABLE-US-00002 Primer forward 1: GCTGCACACTGTAGGAGAAGACTGG (SEQ ID
NO: 19 in the Sequence Listing); and Primer reverse 1:
TGACCGATGGAACGTGGACTC (SEQ ID NO: 20 in the Sequence Listing).
[0292] The DNA polymerase used was KOD-plus-ver. 2 (manufactured by
Toyobo Co., Ltd.). The reaction was performed according to the
instructions for 10 cycles under conditions involving an annealing
temperature of 64.degree. C. and an elongation temperature of
68.degree. C. The PCR product was digested with restriction enzymes
NotI-HF (manufactured by New England Biolabs Inc.) and SfiI
(manufactured by New England Biolabs Inc.), and this fragment was
used as an insert in ligation described later.
[0293] A phagemid vector pCANTAB 5E vector (manufactured by GE
Healthcare Japan Corp. (formerly Pharmacia)) was digested with
restriction enzymes NotI-HF (manufactured by New England Biolabs
Inc.) and SfiI (manufactured by New England Biolabs Inc.).
Subsequently, the resulting fragment of the restriction
enzyme-digested vector was dephosphorylated using Escherichia
coli-derived alkaline phosphatase (manufactured by Takara Bio Inc.)
and then used as a vector in ligation described later.
[0294] The insert and the vector were ligated using T4 DNA ligase
(manufactured by New England Biolabs Inc.) (molar ratio between the
vector and the insert: 1:3). An Escherichia coli TG-1 strain was
transformed with this ligation product by electroporation (using
BTX Electro Cell Manipulator 600 manufactured by BTX Instrument
Division, Harvard Apparatus, Inc.; voltage: 1.98 kV, electric
resistance: 186 ohms), then inoculated onto a plate of an LB medium
containing 100 .mu.g/mL ampicillin and 1% glucose (hereinafter,
referred to as LB/Amp/1% Glu), and cultured at 30.degree. C. for 12
hours to obtain 3.5.times.10.sup.10 Escherichia coli colonies.
3) Large-Scale Preparation of Randomly Mutated TACI_d2 Phage
[0295] From the Escherichia coli colonies thus obtained in the
paragraph 2), an Escherichia coli suspension with OD.sub.600 nm=0.3
was prepared using a 2.times.YT medium containing 100 .mu.g/mL
ampicillin and 1% glucose (hereinafter, referred to as
2.times.YT/Amp/1% Glu). The bacterial cells were shake-cultured at
37.degree. C. and allowed to grow until OD.sub.600 nm=0.5. A
sufficient amount of HYPERPHAGE M13K07 ApIII (manufactured by
Progen Biotechnik GmbH) was added thereto to infect the cells at
37.degree. C. for 30 minutes. Subsequently, 100 .mu.g/mL
ampicillin, 100 .mu.g/mL kanamycin, and 0.25 mM IPTG (hereinafter,
collectively referred to as "2.times.YT/Amp/Kan/0.25 mM IPTG") were
added to the Escherichia coli, and the cells were shake-cultured
overnight at 22.degree. C. 20% polyethylene glycol 6000 and a 2.5 M
NaCl solution (hereinafter, collectively referred to as "20%
PEG/2.5 M NaCl solution") were added to the recovered culture
supernatant, in 1/4 of the amount of the culture supernatant, to
precipitate phages. The precipitated phages were suspended in
phosphate buffered saline (hereinafter, referred to as "PBS") and
used as a randomly mutated TACI-displaying phage library in
experiments below.
[0296] An Escherichia coli TG-1 strain was infected with this phage
solution and inoculated to an LB/Amp/1% Glu plate. The number of
formed colonies was measured. This phage library had a titer of
1.2.times.10.sup.13 phages/mL.
Example 2
[0297] Screening for TACI_d2 Mutant Binding to Target Molecule
1) Liquid-Phase Panning Method
[0298] Each target protein (target molecule) was biotinylated using
EZ-Link NHS-Chromogenic Biotin Reagent (manufactured by Thermo
Fisher Scientific K.K.) according to the instructions. To this
target molecule, a TACI_d2 mutant-displaying phage was added and
reacted overnight or for 2 hours. The TACI_d2 mutant-displaying
phage used was a randomly mutated TACI_d2-displaying phage library
for the 1st round of liquid-phase panning, and phages (randomly
mutated TACI_d2-displaying phage library) prepared from Escherichia
coli colonies obtained in the preceding round, for the 2nd or later
rounds. Dynabeads M-280 Streptavidin (manufactured by Invitrogen
Corp.; hereinafter, referred to as "Dynabeads") was added to this
mixed solution to bind the biotinylated target molecule to the
Dynabeads. The
[0299] Dynabeads were washed with PBS containing 0.05% Tween-20
(hereinafter, referred to as "PBST") a predetermined amount of
times. Then, a TACI_d2 mutant-displaying phage bound with the
biotinylated target molecule on the surface of the Dynabeads was
eluted using 0.1 M glycine-HC1/500 mM NaCl (pH 2.2). The eluted
phage was immediately neutralized with 1 M Tris-HCl (pH 8.0) and
allowed to infect an Escherichia coli TG-1 strain, which was in
turn inoculated to an LB/Amp/1% Glu plate and cultured at
30.degree. C. for 12 hours or longer.
2) Solid-Phase Panning Method
[0300] Each target protein (target molecule) was added to Nunc
Maxisorp flat-bottom 96-well plate (Nunc) (hereinafter, referred to
as a Maxisorp plate) and solid-phase immobilized overnight at
4.degree. C. To the Maxisorp plate, TACI_d2 mutant-displaying
phages were added and reacted for 2 hours. The TACI_d2
mutant-displaying phages used for the 1st round of solid-phase
panning were randomly mutated TACI_d2 mutant-displaying phages, and
phages (randomly mutated TACI_d2-displaying phage library) prepared
from Escherichia coli colonies obtained in the preceding round,
were used for the 2nd or later rounds. The Maxisorp plate was
washed with PBST a predetermined amount of times. Then, a TACI
mutant-displaying phage bound with the target molecule solid-phase
immobilized on the Maxisorp plate was eluted using 0.1 M
glycine-HCl/500 mM NaCl (pH 2.2). The eluted phage was immediately
neutralized with 1 M Tris-HCl (pH 8.0) and allowed to infect an
Escherichia coli TG-1 strain, which was in turn inoculated to an
LB/Amp/1% Glu plate and cultured at 30.degree. C. for 12 hours or
longer.
3) Preparation of Phage for Use in Next Round
[0301] From the Escherichia coli colonies obtained by panning, an
Escherichia coli suspension with OD.sub.600 nm=0.3 for use in a
next round was prepared using 2.times.YT/Amp/1% Glu. The bacterial
cells were shake-cultured at 37.degree. C. and allowed to grow
until OD.sub.600 nm=0.5. A sufficient amount of helper phages
M13K07 was added thereto to infect the cells at 37.degree. C. for
30 minutes. After addition of 2.times.YT/Amp/Kan/0.25 mM IPTG, the
helper phage-infected Escherichia coli was shake-cultured overnight
at 22.degree. C. 20% PEG/2.5 M NaCl solution was added to the
recovered culture supernatant to precipitate phages. The
precipitated phages were suspended in PBS and were used in the next
round of panning.
4) Screening for TACI_d2 Mutant Binding to Target Molecule
[0302] Three or four rounds of liquid-phase or solid-phase panning
were performed using any one of 6 types of recombinant proteins as
a target molecule: IgG-Free, Protease-Free Bovine Serum Albumin
(manufactured by Jackson ImmunoResearch Laboratories, Inc.;
hereinafter, referred to as "BSA"), Recombinant Human EphA2, CF
(manufactured by R&D systems, Inc.; hereinafter, referred to as
"hEphA2"), Recombinant Human EGF R/Fc Chimera, CF (manufactured by
R&D systems, Inc.; hereinafter, referred to as "hEGFR/Fc"),
Recombinant Human ErbB2/Fc Chimera, CF (manufactured by R&D
systems, Inc.; hereinafter, referred to as "hErbB2/Fc"),
Recombinant Human VEGF.sub.165 (manufactured by PeproTech, Inc.;
hereinafter, referred to as "hVEGF"), and Recombinant Human
TNF-.alpha. (manufactured by PeproTech, Inc.; hereinafter, referred
to as "hTNF-.alpha.").
Example 3
[0303] Evaluation of Binding Activity of TACI_d2 Mutant(s) Obtained
by Panning Against Target Molecule
1) Preparation of Phage or Use in Phage ELISA--(1)
[0304] Escherichia coli suspension with OD.sub.600 nm=0.3 was
prepared using 2.times.YT/Amp/1% Glu from the whole Escherichia
coli colonies obtained by the final round of panning. The bacterial
cells were shake-cultured at 37.degree. C. and allowed to grow
until OD.sub.600 nm=0.5. A sufficient amount of helper phages
M13K07 was added thereto to infect the cells at 37.degree. C. for
30 minutes. After addition of 2.times.YT/Amp/Kan/0.25 mM IPTG, the
helper phage-infected Escherichia coli was shake-cultured overnight
at 22.degree. C. 20% PEG/2.5 M NaCl solution was added to the
recovered culture supernatant to precipitate phages, which were
then suspended in PBS. This phage stock solution and a series of
two-fold dilutions with PBS were used in phage ELISA.
2) Preparation of Phage for Use in Phage ELISA--(2)
[0305] One single colony selected from the Escherichia coli
colonies obtained by the final round of panning was inoculated to
2.times.YT/Amp/1% Glu. The bacterial cells were shake-cultured at
37.degree. C. and allowed to grow until OD.sub.600 nm=0.5. A
sufficient amount of helper phages M13K07 was added thereto to
infect the cells at 37.degree. C. for 30 minutes. After addition of
2.times.YT/Amp/Kan/0.25 mM IPTG, the helper phage-infected
Escherichia coli was shake-cultured overnight at 22.degree. C. 20%
PEG/2.5 M NaCl solution was added to the recovered culture
supernatant to precipitate phages. The precipitated phages were
suspended in PBS. This phage stock solution and a series of
two-fold dilutions with PBS were used in phage ELISA.
3) Phage ELISA
[0306] Each target molecule or each negative control protein
(hEphA2 for BSA used as a target molecule, and BSA for any of the
other proteins used as a target molecule) was added to a Maxisorp
plate and solid-phase immobilized overnight at 4.degree. C. The
TACI mutant-displaying phages prepared in the paragraphs 1) and 2)
of Example 2 were added to this Maxisorp plate with the solid-phase
immobilized target protein or negative control protein, and reacted
for 2 hours. The Maxisorp plate was washed with Tris buffered
saline containing 0.05% Tween-20 (hereinafter, referred to as
TBST). Then, HRP/Anti-M13 Monoclonal Conjugate (manufactured by GE
Healthcare Japan Corp.) (hereinafter, referred to as an "Anti-M13
antibody") diluted 5000-fold was added thereto. After washing with
TBST again, the amount of a phage bound with the target molecule
was detected as absorbance at a wavelength of 405 nm using ELISA
POD Substrate A.B.T.S. Kit (manufactured by Nacalai Tesque, Inc.)
(hereinafter, referred to as "ELISA POD").
[0307] As shown in FIGS. 1(A), 1(B), and 1(C), TACI mutant(s)
binding to each target molecule was screened for or enriched from
the randomly mutated TACI_d2 library by the panning operation.
Example 4
[0308] Comparison of specificity between wild-type TACI_d2 and
hEphA2-binding TACI_d2 mutant
1) Preparation of Phage for Use in Phage ELISA
[0309] As to 3 clones (.alpha.-EphA2 #1 to #3: corresponding to SEQ
ID NOs: 3 to 5, respectively, in the Sequence Listing and to Nos. 2
to 4, respectively in FIG. 4) selected from the Escherichia coli
colonies after the final round of panning with hEphA2 as a target
molecule, an Escherichia coli suspension with OD.sub.600 nm=0.3 was
prepared using 2.times.YT/Amp/1% Glu. The Escherichia coli cells of
each clone were shake-cultured at 37.degree. C. and allowed to grow
until OD.sub.600 nm=0.5. A sufficient amount of helper phages
M13K07 was added thereto to infect the cells at 37.degree. C. for
30 minutes. After addition of 2.times.YT/Amp/Kan/0.25 mM IPTG, the
helper phage-infected Escherichia coli was shake-cultured overnight
at 22.degree. C. 20% PEG/2.5 M NaCl solution was added to the
recovered culture supernatant to precipitate phages. The
precipitated phages were suspended in PBS. This phage stock
solution and a series of two-fold dilutions with PBS were used in
phage ELISA. An Escherichia coli TG-1 strain having a pCANTAB 5E
vector containing the wild-type TACI_d2 gene was also subjected to
the same operation as above to prepare phages.
2) Phage ELISA
[0310] Each of hEphA2, BSA, Recombinant Human BAFF/BLyS/TNFSF13B
(R&D systems, Inc.; a polypeptide consisting of amino acids
from Ala at position 134 to Leu at position 285 in human BAFF
(UniProtKB/Swiss-Prot Accession #Q9Y275) and containing an
N-terminally linked histidine tag, etc.; hereinafter referred to as
"hBAFF"), and Recombinant Mouse EphA2/Fc Chimera, CF (R&D
systems, Inc.; a polypeptide consisting of amino acids from Ala at
position 22 to Ala at position 535 in mouse EphA2 (NCBI Accession
No. #AAA82113) and containing a C-terminally linked Fc region (from
Pro at position 100 to Lys at position 330) of human immunoglobulin
G1 (hereinafter, referred to as "hIgG1"); hereinafter, referred to
as "mEphA2/Fc") was added to a Maxisorp plate and solid-phase
immobilized overnight at 4.degree. C. TACI_d2 mutant-displaying
phages were added to these Maxisorp plates and reacted for 2 hours.
Each Maxisorp plate was washed with TBST. Then, an Anti-M13
antibody diluted 5000-fold was added thereto. After washing of the
Maxisorp plate with TBST again, the amount of a phage bound with
the protein solid-phase immobilized on the plate was detected as
absorbance at a wavelength of 405 nm using ELISA POD.
[0311] The results are shown in FIGS. 2(A) and 2(B). .alpha.-EphA2
#1 to #3 were obtained as TACI_d2 mutants binding to hEphA2. All of
these U-EphA2 #1 to #3 lost the ability of wild-type TACI_d2 to
bind to the endogenous ligand hBAFF. .alpha.-EphA2 #2 and #3
exhibited cross reactivity to mEphA2 at a level equivalent to
wild-type TACI_d2, whereas .alpha.-EphA2 #1 had stronger cross
reactivity.
Example 5
[0312] Confirmation of Binding of hEphA2-Binding TACI_d2 Mutant to
Human EphA2-Expressing Cell
1) Cell Culture and Medium
[0313] Human embryonic kidney cells (HEK293T cells) were cultured
at 37.degree. C. in the presence of 5% CO.sub.2 using a Dulbecco's
modified eagle's medium (hereinafter, referred to as "DMEM";
manufactured by GIBCO, Life Technologies Corp.) containing 10%
fetal bovine serum (FBS) (hereinafter, this medium is referred to
as "DMEM-10% FBS"). For use in transfection or flow cytometry, the
cells were dissociated from the plate for culture using 0.05%
trypsin-EDTA (GIBCO), and the cell suspension was then centrifuged
to recover cells, which were then resuspended in DMEM-10%FBS and
used.
2) Transfection
[0314] The HEK293T cells were transfected with a pcDNA-DEST40
Gateway vector (manufactured by Invitrogen Corp.) having an insert
of the human EphA2 gene or the human ErbB2 gene as a negative
control using Lipofectamine 2000 (manufactured by Invitrogen Corp.)
according to the instructions.
3) Preparation of Cell
[0315] The transfected cells were recovered. The recovered cells
were suspended in a FACS buffer (PBS containing 5% FBS) and applied
to a nylon mesh. The solution was dispensed in an amount of
5.times.10.sup.5 cells/well to 96 Well Cell Culture Cluster Round
Bottom With Lid (manufactured by Costar, Corning Inc.) and
centrifuged to remove a supernatant. The phage stock solution
prepared in the paragraph 1) of Example 4 was added to the
resulting cells at a concentration of 50 .mu.L/well, and the
mixture was left standing at 4.degree. C. for 30 minutes. A FACS
buffer was added thereto at a concentration of 150 .mu.L/well, and
the mixture was centrifuged to remove a supernatant. Again, a FACS
buffer was added thereto at a concentration of 200 .mu.L/well, and
the mixture was centrifuged to remove a supernatant (hereinafter,
cells were washed in the same way as this operation). An Anti-M13
antibody diluted 100-fold was added to the obtained cells at a
concentration of 50 .mu.L/well as a primary antibody, and the
mixture was left standing at 4.degree. C. for 30 minutes, followed
by washing of the cells. Subsequently, FLUORESCEIN-CONJUGATED GOAT
IGG FRACTION TO MOUSE IGG (manufactured by Cappel, MP Biomedicals,
LLC) diluted 1000-fold was added thereto as a secondary antibody,
and the mixture was left standing at 4.degree. C. for 30 minutes,
followed by washing of the cells. The obtained cells were suspended
in 250 .mu.L of a FACS buffer and subjected to Cytomics FC 500 Flow
Cytometry System (manufactured by Beckman Coulter, Inc.).
Fluorescently stained cells were detected.
[0316] The results are shown in FIGS. 3(A) and 3(B). .alpha.-EphA2
#1 and #2 bound to not only recombinant hEphA2 but human EphA2
expressed on cell surface.
Example 6
[0317] Sequencing of TACI_d2 mutants binding to various target
molecules
[0318] As to TACI_d2 mutant peptides confirmed to bind to various
target molecules in the same way as in the paragraphs 2) and 3) of
Example 3, Escherichia coli expressing each of the peptides was
isolated and cultured overnight at 37.degree. C. using a 2 x YT
medium. A pCANTAB 5E vector having an insert of a gene encoding the
peptide was prepared from the recovered Escherichia coli using
QIAGEN Plasmid Mini Kit (manufactured by QIAGEN N.V.). The vector
was sequenced using a pCANTAB-S1 primer
(5'-CAACGTAAAAAATTATTATTCGC-3': SEQ ID NO: 21 in the Sequence
Listing).
[0319] The hBAFF described above was used as an endogenous
TACI-binding molecule.
(1) Epidermal Growth Factor Receptor (EGFR)
[0320] A TACI_d2 mutant binding to the extracellular domain of
human EGFR was screened for, and the obtained peptides were
analyzed for their amino acid sequences.
[0321] The human EGFR used was a fusion protein (hEGFR/Fc described
above) of the EGFR extracellular domain (from Leu at position 25 to
Ser at position 645 in UniProtKB/Swiss-Prot Accession #P00533) and
an hIgG1 Fc region (from Pro at position 100 to Lys at position
330).
[0322] The amino acid sequences of the obtained 3 peptides are
shown in SEQ ID NOs: 6 to 8 in the Sequence Listing (Nos. 5 to 7 in
FIG. 4).
(2) Vascular Endothelial Growth Factor (VEGF)
[0323] The VEGF used was a human isoform VEGF165 (CAS Registry File
Registry Number 1217406-67-1; hVEGF described above).
[0324] A TACI_d2 mutant binding to human VEGF was screened for, and
the obtained 6 peptides were analyzed for their amino acid
sequences. As a result, the amino acid sequences were as shown in
SEQ ID NOs: 9 to 14 in the Sequence Listing (Nos. 8 to 13 in FIG.
4).
(3) Tumor Necrosis Factor .alpha. (TNF-.alpha.)
[0325] The TNF-.alpha. used was human TNF (CAS Registry File
Registry Number 1228062-30-3; hTNF-.alpha. described above).
[0326] A TACI_d2 mutant binding to human TNF.alpha. was screened
for, and the obtained 3 peptides were analyzed for their amino acid
sequences. As a result, the amino acid sequences were as shown in
SEQ ID NOs: 15 to 17 in the Sequence Listing (Nos. 14 to 16 in FIG.
4). (4) In all of the target molecule-binding TACI_d2 mutants
obtained in the preceding paragraphs (1) to (3), Phe at position 78
within the TACI_d2 structure, i.e., at position 11 of SEQ ID NO: 1
in the Sequence Listing, was substituted by a different amino
acid.
Example 7
[0327] Determination of Dissociation Constant of VEGF-Binding
TACI_d2 Mutant for VEGF
[0328] The gene of the hVEGF-binding TACI_d2 mutant (No. 9 in FIG.
4: SEQ ID NO: 10 in the Sequence Listing) obtained in Example 2 was
amplified by PCR and inserted to a vector pET-32a for expression in
Escherichia coli (Novagen, Merck KGaA). An Escherichia coli
Origami(TM) B strain (Merck KGaA) was transformed with this vector
and shake-cultured at 37.degree. C. until OD.sub.600 nm=0.7. IPTG
was added thereto at a final concentration of 1 mM to induce the
expression of the mutant. Then, the bacterium was shake-cultured at
16.degree. C. for 12 hours or longer. The cultured Escherichia coli
was disrupted by ultrasonication and centrifuged. The obtained
supernatant was applied to a HisTALON(TM) column (Clontech
Laboratories, Inc.) for purification. Since the expressed mutant
was thioredoxin-tagged and His-tagged, these tags were removed by
cleavage using a Thrombin cleavage capture kit and application to a
HisTALON(TM) column again. The purified mutant was subjected to
size exclusion chromatography (Superdex 75 HR 10/30 column, GE
Healthcare Japan Corp.). A peak corresponding to the molecular
weight of the mutant (monomer) was recovered and subjected to
affinity analysis. The affinity of the mutant was determined by a
surface plasmon resonance method using Biacore 3000 (GE Healthcare
Japan Corp.). The mutant was applied at various concentrations to
hVEGF immobilized on a sensor chip. The dissociation constant of
the mutant for hVEGF was determined from the obtained sensorgram.
As a result, the mutant had a dissociation constant of 14 .mu.M for
hVEGF.
INDUSTRIAL APPLICABILITY
[0329] The peptide library of the present invention is useful in
screening for a peptide binding to a target molecule.
FREE TEXT OF SEQUENCE LISTING
[0330] SEQ ID NO: 3--Amino acid sequence of human EphA2-binding
peptide .alpha.-EphA2 #1 [0331] SEQ ID NO: 4--Amino acid sequence
of human EphA2-binding peptide .alpha.-EphA2 #2 [0332] SEQ ID NO:
5--Amino acid sequence of human EphA2-binding peptide .alpha.-EphA2
#3 [0333] SEQ ID NO: 6--Amino acid sequence of human EGFR-binding
peptide (1) [0334] SEQ ID NO: 7--Amino acid sequence of human
EGFR-binding peptide (2) [0335] SEQ ID NO: 8--Amino acid sequence
of human EGFR-binding peptide (3) [0336] SEQ ID NO: 9--Amino acid
sequence of human VEGF-binding peptide (1) [0337] SEQ ID NO:
10--Amino acid sequence of human VEGF-binding peptide (2) [0338]
SEQ ID NO: 11--Amino acid sequence of human VEGF-binding peptide
(3) [0339] SEQ ID NO: 12--Amino acid sequence of human VEGF-binding
peptide (4) [0340] SEQ ID NO: 13--Amino acid sequence of human
VEGF-binding peptide (5) [0341] SEQ ID NO: 14--Amino acid sequence
of human VEGF-binding peptide (6) [0342] SEQ ID NO: 15--Amino acid
sequence of human=TNF.alpha.-binding peptide (1) [0343] SEQ ID NO:
16--Amino acid sequence of human=TNF.alpha.-binding peptide (2)
[0344] SEQ ID NO: 17--Amino acid sequence of
human=TNF.alpha.-binding peptide (3) [0345] SEQ ID NO:
18--Nucleotide sequence of randomly mutated TACI_d2 oligonucleotide
[0346] SEQ ID NO: 19--Nucleotide sequence of PCR primer Primer
Forward 1 [0347] SEQ ID NO: 20--Nucleotide sequence of PCR primer
Primer Reverse 1 [0348] SEQ ID NO: 21--Nucleotide sequence of
pCANTAB-S1 primer as primer for sequencing
Sequence CWU 1
1
21139PRTArtificial SequenceAmino acid sequence of human TACI_d2
peptideMISC_FEATURE(11)..(11)Any amino
acidMISC_FEATURE(13)..(17)Any amino acidMISC_FEATURE(20)..(20)Any
amino acidMISC_FEATURE(25)..(25)Any amino
acidMISC_FEATURE(28)..(28)Any amino acidMISC_FEATURE(31)..(32)Any
amino acid 1Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Xaa Tyr Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Asp Cys Xaa Ser Cys Ala Ser Xaa Cys Gly Xaa
His Pro Xaa Xaa 20 25 30 Cys Ala Tyr Phe Cys Glu Asn 35
238PRTArtificial SequenceAmino acid sequence of human BSA-binding
peptide 2Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Gln Tyr Trp Arg
Glu Lys 1 5 10 15 Met Asp Cys Glu Cys Ala Ser Lys Cys Gly Asn His
Pro Asp Ile Cys 20 25 30 Ala Tyr Phe Cys Glu Asn 35
339PRTArtificial SequenceAmino acid sequence of human EphA2-binding
peptide a-EphA2 #1 3Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Gln Tyr
Leu Leu Arg Glu 1 5 10 15 Trp Asp Cys Asp Ser Cys Ala Ser Glu Cys
Gly Ser His Pro His Tyr 20 25 30 Cys Ala Tyr Phe Cys Glu Asn 35
439PRTArtificial SequenceAmino acid sequence of human EphA2-binding
peptide a-EphA2 #2 4Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Met Tyr
Leu Leu Lys Glu 1 5 10 15 Trp Asp Cys Ala Ser Cys Ala Ser Ala Cys
Gly Asn His Pro His Tyr 20 25 30 Cys Ala Tyr Phe Cys Glu Asn 35
539PRTArtificial SequenceAmino acid sequence of human EphA2-binding
peptide a-EphA2 #3 5Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys His Tyr
Leu Leu Lys Glu 1 5 10 15 Tyr Asp Cys Asp Ser Cys Ala Ser Glu Cys
Gly Tyr His Pro Asp Tyr 20 25 30 Cys Ala Tyr Phe Cys Glu Asn 35
639PRTArtificial SequenceAmino acid sequence of human EGFR-binding
peptide (1) 6Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys Ser Tyr Gly
Ala Ile Met 1 5 10 15 Tyr Asp Cys Ser Ser Cys Ala Ser Tyr Cys Gly
Glu His Pro Trp His 20 25 30 Cys Ala Tyr Phe Cys Glu Asn 35
739PRTArtificial Sequence- Amino acid sequence of human
EGFR-binding peptide (2) 7Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Glu Tyr Gly Ala Ile Ala 1 5 10 15 Trp Asp Cys Ser Ser Cys Ala Ser
Tyr Cys Gly Ala His Pro Phe Glu 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 839PRTArtificial SequenceAmino acid sequence of human
EGFR-binding peptide (3) 8Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Asn Tyr Ile His Gln Gln 1 5 10 15 Trp Asp Cys Ala Ser Cys Ala Ser
Glu Cys Gly Gly His Pro Asn Tyr 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 939PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (1) 9Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Trp Tyr Met Thr Trp Glu 1 5 10 15 Ser Asp Cys Lys Ser Cys Ala Ser
Trp Cys Gly Ser His Pro Phe Asp 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1039PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (2) 10Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Met Tyr Asp Leu Tyr Gly 1 5 10 15 Phe Asp Cys Arg Ser Cys Ala Ser
Met Cys Gly Lys His Pro Asp Leu 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1139PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (3) 11Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Met Tyr Met Val Trp Thr 1 5 10 15 Gln Asp Cys Lys Ser Cys Ala Ser
Trp Cys Gly Ala His Pro Val Ala 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1239PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (4) 12Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Ile Tyr Asn Gln Tyr Gly 1 5 10 15 Phe Asp Cys Lys Ser Cys Ala Ser
Trp Cys Gly Lys His Pro Asp Met 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1339PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (5) 13Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Ile Tyr Met Thr Trp His 1 5 10 15 Asp Asp Cys His Ser Cys Ala Ser
Leu Cys Gly Ser His Pro Leu Phe 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1439PRTArtificial SequenceAmino acid sequence of human
VEGF-binding peptide (6) 14Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Asp Tyr Met Val Phe Gly 1 5 10 15 Gln Asp Cys His Ser Cys Ala Ser
Trp Cys Gly Lys His Pro Val Ala 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1539PRTArtificial SequenceAmino acid sequence of human
TNFa-binding peptide (1) 15Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Gln Tyr Met Ala Gly His 1 5 10 15 Phe Asp Cys Asn Ser Cys Ala Ser
Arg Tyr Gly His His Pro Leu Met 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1639PRTArtificial SequenceAmino acid sequence of human
TNFa-binding peptide (2) 16Ser Leu Ser Cys Arg Lys Glu Gln Asp Lys
Thr Tyr Ile Glu Tyr Gly 1 5 10 15 Phe Asp Cys Arg Ser Cys Ala Ser
Gly Cys Gly Gly His Pro Leu Met 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 1739PRTArtificial SequenceAmino acid sequence of human
TNFa-binding peptide (3) 17Ser Leu Ser Cys Arg Lys Glu Gln Gly Lys
Ser Tyr Thr Ser Glu Trp 1 5 10 15 Phe Asp Cys Ala Ser Cys Ala Ser
Lys Tyr Gly Lys His Pro Leu Val 20 25 30 Cys Ala Tyr Phe Cys Glu
Asn 35 18183DNAArtificial SequenceNucleotide sequence of randomly
mutated TACI_d2 oligonucleotidemisc_feature(68)..(70)n is a, c, g,
or tmisc_feature(74)..(88)n is a, c, g, or tmisc_feature(95)..(97)n
is a, c, g, or tmisc_feature(110)..(112)n is a, c, g, or
tmisc_feature(119)..(121)n is a, c, g, or
tmisc_feature(128)..(133)n is a, c, g, or t 18gctgcacact gtaggagaag
actgggccca gccggccagc ctgagttgcc gtaaagaaca 60gggcaagnnn tatnnnnnnn
nnnnnnnnga ctgcnnnagc tgcgcgagcn nntgtggann 120ncatcctnnn
nnntgcgcgt atttttgcga aaacgcggcc gcgagtccac gttccatcgg 180tca
1831925DNAArtificial SequenceNucleotide sequence of PCR primer
Primer Forward 1 19gctgcacact gtaggagaag actgg 252021DNAArtificial
SequenceNucleotide sequence of PCR primer Primer Reverse 1
20tgaccgatgg aacgtggact c 212123DNAArtificial SequenceNucleotide
sequence of pCANTAB-S1 primer as primer for sequencing 21caacgtaaaa
aattattatt cgc 23
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