U.S. patent application number 14/528197 was filed with the patent office on 2015-02-19 for method for culturing pluripotent stem cell, and polypeptide to be used therefor.
This patent application is currently assigned to FUJIFILM CORPORATION. The applicant listed for this patent is FUJIFILM CORPORATION. Invention is credited to Yoshihide IWAKI, Rie IWATA, Yuta MURAKAMI, Tasuku SASAKI.
Application Number | 20150050737 14/528197 |
Document ID | / |
Family ID | 49514369 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150050737 |
Kind Code |
A1 |
MURAKAMI; Yuta ; et
al. |
February 19, 2015 |
METHOD FOR CULTURING PLURIPOTENT STEM CELL, AND POLYPEPTIDE TO BE
USED THEREFOR
Abstract
A polypeptide including: (1) a first region containing at least
one selected from the group consisting of an amino acid sequence
represented by CSYYQSC (SEQ ID NO:1) and an amino acid sequence
represented by RGD; and (2) a second region containing (2-i) an
amino acid sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN
(SEQ ID NO:2), (2-ii) an amino acid sequence having an identity of
not less than 50% to the amino acid sequence represented by SEQ ID
NO:2 and having an adsorption ability to a cultivation container,
or (2-iii) an amino acid sequence that is the amino acid sequence
represented by SEQ ID NO:2 in which from 1 to 30 amino acid
residues are added, substituted, or deleted, and has an adsorption
ability to a cultivation container, in which the polypeptide
includes from 40 to 450 amino acid residues.
Inventors: |
MURAKAMI; Yuta; (Kanagawa,
JP) ; IWATA; Rie; (Kanagawa, JP) ; IWAKI;
Yoshihide; (Kanagawa, JP) ; SASAKI; Tasuku;
(Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FUJIFILM CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
FUJIFILM CORPORATION
Tokyo
JP
|
Family ID: |
49514369 |
Appl. No.: |
14/528197 |
Filed: |
October 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2013/062122 |
Apr 24, 2013 |
|
|
|
14528197 |
|
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Current U.S.
Class: |
435/402 ;
530/300; 530/350 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2533/50 20130101; C07K 14/001 20130101; C07K 14/47 20130101;
C12N 2537/00 20130101; C12N 5/0607 20130101; C07K 14/78 20130101;
C12N 5/0068 20130101; C12N 2533/52 20130101 |
Class at
Publication: |
435/402 ;
530/300; 530/350 |
International
Class: |
C12N 5/074 20060101
C12N005/074; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 2012 |
JP |
2012-104816 |
Claims
1. A polypeptide consisting of from 40 to 450 amino acid residues
and comprising: (1) a first region comprising at least one selected
from the group consisting of an amino acid sequence represented by
CSYYQSC (SEQ ID NO:1) and an amino acid sequence represented by
RGD; and (2) a second region comprising (2-i) an amino acid
sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID
NO:2), (2-ii) an amino acid sequence having an identity of not less
than 50% to an amino acid sequence represented by SEQ ID NO:2 and
having an adsorption ability to a cultivation container, or (2-iii)
an amino acid sequence that is the amino acid sequence represented
by SEQ ID NO:2, in which from 1 to 30 amino acid residues are
added, substituted, or deleted, and that has an adsorption ability
to a cultivation container.
2. The polypeptide according to claim 1, wherein a GRAVY value is
from -2.0 to -0.95.
3. The polypeptide according to claim 1, wherein the first region
comprises both of the amino acid sequence represented by CSYYQSC
(SEQ ID NO:1) and the amino acid sequence represented by RGD.
4. The polypeptide according to claim 1, wherein the polypeptide
consists of from 40 to 400 amino acid residues.
5. The polypeptide according to claim 1, further comprising a third
region consisting of any one amino acid sequence of the following
(3-i) to (3-iii): (3-i) an amino acid sequence consisting of the
56th to 341st amino acid residues in an amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; (3-ii) an amino acid sequence having an identity of not
less than 50% to the (3-i) amino acid sequence or partial amino
acid sequence thereof; or (3-iii) an amino acid sequence that is
the (3-i) amino acid sequence or partial amino acid sequence
thereof in which from 1 to 30 amino acid residues are added,
substituted, or deleted.
6. The polypeptide according to claim 1, further comprising a third
region consisting of any one amino acid sequence of the following
(3a-i) to (3a-iii): (3a-i) an amino acid sequence consisting of the
132th to 341st amino acid residues in an amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; (3a-ii) an amino acid sequence having an identity of not
less than 50% to the (3a-i) amino acid sequence or partial amino
acid sequence thereof; or (3a-iii) an amino acid sequence that is
the (3a-i) amino acid sequence or partial amino acid sequence
thereof in which from 1 to 30 amino acid residues are added,
substituted, or deleted.
7. The polypeptide according to claim 1, further comprising a third
region consisting of any one amino acid sequence of the following
(3b-i) to (3b-iii): (3b-i) an amino acid sequence consisting of the
269th to 341st amino acid residues in an amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; (3b-ii) an amino acid sequence having an identity of not
less than 50% to the (3b-i) amino acid sequence or partial amino
acid sequence thereof; or (3b-iii) an amino acid sequence that is
the (3b-i) amino acid sequence or partial amino acid sequence
thereof in which from 1 to 30 amino acid residues are added,
substituted, or deleted.
8. The polypeptide according to claim 1, further comprising a
fourth region consisting of any one amino acid sequence of the
following (4-i) to (4-iii): (4-i) an amino acid sequence consisting
of the 374th to 459th amino acid residues in an amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; (4-ii) an amino acid sequence having an identity of not
less than 50% to the (4-i) amino acid sequence or partial amino
acid sequence thereof; or (4-iii) an amino acid sequence that is
the (4-i) amino acid sequence or partial amino acid sequence
thereof in which from 1 to 30 amino acid residues are added,
substituted, or deleted.
9. The polypeptide according to claim 1, wherein the (2-ii) amino
acid sequence has an identity of not less than 80% to the amino
acid sequence represented by SEQ ID NO:2.
10. The polypeptide according to claim 1, wherein the (2-iii) amino
acid sequence is the amino acid sequence represented by SEQ ID NO:2
in which from 1 to 15 amino acid residues are added, substituted,
or deleted.
11. A polypeptide consisting of from 80 to 450 amino acid residues
and comprising: (1) a first region consisting of an amino acid
sequence consisting of the 25th to 47th amino acid residues in an
amino acid sequence represented by SEQ ID NO:3; (2) a second region
consisting of an amino acid sequence consisting of the 342nd to
373rd amino acid residues in the amino acid sequence represented by
SEQ ID NO:3; and at least one selected from the group consisting of
the following third and fourth regions: (3) the third region
consisting of an amino acid sequence consisting of the 269th to
341st amino acid residues in the amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof; and (4) the
fourth region consisting of an amino acid sequence consisting of
the 374th to 459th amino acid residues in the amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof.
12. A polypeptide consisting of from 100 to 450 amino acid residues
and comprising: (1) a first region consisting of an amino acid
sequence consisting of the 1st to 55th amino acid residues in an
amino acid sequence represented by SEQ ID NO:3; (2) a second region
consisting of an amino acid sequence consisting of the 342nd to
373rd amino acid residues in the amino acid sequence represented by
SEQ ID NO:3; and at least one selected from the group consisting of
the following third and fourth regions: (3) the third region
consisting of an amino acid sequence consisting of the 269th to
341st amino acid residues in the amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof; and (4) the
fourth region consisting of an amino acid sequence consisting of
the 374th to 459th amino acid residues in the amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof.
13. The polypeptide according to claim 11, wherein the polypeptide
has a GRAVY value of from -2.0 to -0.95.
14. The polypeptide according to claim 1, the number of amino acid
residues of which being 250 or less.
15. The polypeptide according to claim 5, wherein the polypeptide
comprises the third region, and the third region comprises an amino
acid residue other than a cysteine residue at a position
corresponding to a cysteine residue in the amino acid sequence
represented by SEQ ID NO:3.
16. The polypeptide according to claim 5, wherein the polypeptide
comprises the third region, and the third region comprises a serine
residue, an alanine residue, or a glycine residue at a position
corresponding to a cysteine residue in the amino acid sequence
represented by SEQ ID NO:3.
17. The polypeptide according to claim 1, wherein the first region
is located at an N-terminal side of the second region.
18. The polypeptide according to claim 1, wherein two cysteine
residues in the amino acid sequence represented by SEQ ID NO:1 are
cross-linked to each other.
19. A polypeptide comprising an amino acid sequence represented by
any of SEQ ID NO:4 to SEQ ID NO:23, SEQ ID NO:38, or SEQ ID
NO:39.
20. A method of culturing a pluripotent stem cell, comprising:
applying the polypeptide according to claim 1 to a cell culture
surface of a support, to obtain a polypeptide-coated culture
surface; and seeding a pluripotent stem cell on the
polypeptide-coated culture surface and culturing the pluripotent
stem cell.
21. The method of culturing a pluripotent stem cell according to
claim 20, wherein the pluripotent stem cell is at least one
selected from the group consisting of embryonic stem cells, induced
pluripotent stem cells, somatic stem cells, fertilized egg inner
cell mass cells, and early embryonic cells.
22. The method of culturing a pluripotent stem cell according to
claim 20, wherein the pluripotent stem cell is cultured in the
absence of a component derived from a heterologous animal and a
component derived from serum.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of
International Application No. PCT/JP2013/062122, filed Apr. 24,
2013, which is incorporated herein by reference. Further, this
application claims priority from Japanese Patent Application No.
2012-104816, filed May 1, 2012, which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method of culturing a
pluripotent stem cell and to a polypeptide to be used in the
method.
BACKGROUND ART
[0003] Various kinds of regenerative medicine have been developed
for the purpose of the functional recovery of damaged tissues and
the like. Especially, many technologies for primate, particularly
human totipotent or pluripotent stem cells, of which the ultimate
goal have been the regeneration of tissues per se, and the like,
have been reported. Unlike embryonic stem cells, induced
pluripotent stem cells (iPS cells) particularly have had an
advantage that the induced pluripotent stem cells are less
ethically problematic because the induced pluripotent stem cells
are induced from somatic cells.
[0004] In a case of culturing primate totipotent or pluripotent
stem cells (both cells are generically simply referred to as
"pluripotent stem cell" in the invention), it is required to
maintain the primate totipotent or pluripotent stem cells in an
undifferentiated state for a long term. In order to culture the
pluripotent stem cells in an undifferentiated state for a long
term, feeder cells such as mouse fibroblasts are generally
used.
[0005] However, it has been pointed out that the use of the feeder
cells derived from heterologous animals, such as the mouse
fibroblasts, cause contamination of culture liquids with foreign
substances such as antigenic substances derived from heterologous
animals. In a case in which totipotent or pluripotent stem cells
are used in medical applications or applications equivalent
thereto, it has been demanded that the cells are cultured in the
absence of the feeder cells.
[0006] In view of such circumstances, cell-adhesive materials of
which the functions substitute for those of feeder cells have been
developed. For example, it is disclosed in Nature Biotechnology,
2001, Vol. 19, pp. 971-974 that human embryonic stem cells
maintained in an undifferentiated state are successfully cultured
using, as a substitute for feeder cells, Matrigel, which is a
component extracted from mouse sarcoma.
[0007] In Japanese Patent Application Laid-Open (JP-A) No.
2001-17183, a cellular composition that does not contain any feeder
cells but contains proliferating primate progenitor cells is
disclosed, and the cellular composition that further contains an
extracellular matrix is disclosed as a preferred embodiment. In
Japanese Patent Application Laid-Open (JP-A) No. 2010-29186, a cell
culture matrix in which a cell culture surface subjected to plasma
polymerization is further coated with a coating solution containing
a predetermined concentration of extracellular matrix protein and
an aqueous solvent is disclosed, and it is described that the cell
culture matrix has favorable adhesiveness helpful for avoiding the
differentiation of embryonic stem cells. In Japanese National-Phase
Publication (JP-A) No. 2012-502664, a peptide that binds to a
glycosaminoglycan (GAG) is disclosed.
[0008] In Biomaterials, 2010, November; Vol. 31 (32), pp. 8281-8288
and Nature Biotechnology, 2010, Vol. 28, No. 6, pp. 606-610,
recombinant or synthetic peptides containing a vitronectin partial
sequence that can contribute to the long-term culture of embryonic
stem cells, specifically, each of the sequence of 1st to the 52nd
amino acids of natural vitronectin (see Biomaterials, 2010,
November; Vol. 31 (32), pp. 8281-8288) and the sequence of the 41st
to 52nd amino acids containing an RGD sequence (Nature
Biotechnology, 2010, Vol. 28, No. 6, pp. 606-610) are disclosed.
The peptides have been known to be excellent in view of the fact
that: the possibility of contamination with antigenic substances
and the like can be avoided since the peptides are non-biological
samples; and the peptides can be industrially produced.
SUMMARY OF INVENTION
Technical Problem
[0009] However, uses of heterologous feeder cells such as
fibroblasts derived from mice and the like, and components derived
from heterologous animals, including Matrigel derived from mice,
should be avoided as much as possible, considering that pluripotent
stem cells are used in medical applications such as regenerative
medicine or applications equivalent thereto. Even in a case of a
cell or a component derived from a homologous animal, it is
impossible to completely eliminate the possibility of the
contamination of the cell or the component with an antigenic
substance or the like. Any homologous or heterologous material
derived from a living body is not preferred even from an industrial
viewpoint that, for example, the amount of the extracted material
is an extremely minute amount, or the feature of the material may
vary depending on a donor. In recent years, the culture of stem
cells, aimed at being applied to medical applications, under a
chemically identified condition, in which a component or an
antigenic substance derived from a heterologous animal is not
contaminated, has been examined vigorously. However, a material
that can substitute for feeder cells exhibiting a cell culture
feature sufficient for practical applications has not been
found.
[0010] For example, an example of culturing embryonic stem cells
for a long term using recombinant or synthetic peptides containing
a partial sequence of human vitronectin is disclosed as a technique
for avoiding the use of a material per se derived from a living
body in Biomaterials, 2010, November; Vol. 31 (32), pp. 8281-8288
and Nature Biotechnology, 2010, Vol. 28, No. 6, pp. 606-610.
[0011] However, since such peptides have low adsorption to a
cultivation container without discrimination, a step of chemically
binding the peptides to the cultivation container is required. For
example, an introduction of an acrylate into a cell culture surface
in a cultivation container to covalently bind peptides is disclosed
in Nature Biotechnology, 2010, Vol. 28, No. 6, pp. 606-610.
However, this method has poor general-purpose properties and
simpleness since it is impossible to freely select the culture
surface to which the peptides are bound, and only the peptides are
not sufficient for a cell culture feature for culturing embryonic
stem cells (ESCs) and induced pluripotent stem cells (iPS). In JP-A
No. 2012-502664, an isolated peptide binding to a glycosaminoglycan
(GAG) is disclosed, and it is described that the peptide is used in
the long-term culture and maintenance of ESCs and iPS utilizing a
site binding to the glycosaminoglycan. However, only the binding
site to the GAG is not sufficient for a cell culture feature for
culturing ESCs and iPS.
[0012] Accordingly, an object of the invention is to provide a
polypeptide that enables a pluripotent stem cell to proliferate in
an undifferentiated state, does not require treatment of
immobilization thereof to a cultivation container by chemical
bonding, and can be industrially produced, and to provide a method
of culturing a pluripotent stem cell using the polypeptide.
Solution to Problem
[0013] The present inventors repeated extensive research in order
to develop a recombinant protein that enables pluripotent stem
cells to proliferate in an undifferentiated state, has excellent
adsorptivity to a cultivation container, and does not require
treatment of immobilization to a cultivation container by chemical
bonding, and found that a specific polypeptide that comprises an
amino acid residue containing a specific human vitronectin
N-terminal partial sequence and having an adsorption ability to a
cultivation container enables pluripotent stem cells to proliferate
in a culture liquid that does not contain any component derived
from a heterologous animal while maintaining an undifferentiated
state for a long term, and is adsorbed to a cultivation container
without chemical bonding. It was further surprisingly found that
the polypeptide consisting of the sequence has significantly
excellent proliferation properties, compared to recombinant
vitronectin consisting of a human vitronectin full-length sequence,
and the invention was thus accomplished.
[0014] The invention provides the following embodiments:
[0015] [1] A polypeptide consisting of from 40 to 450 amino acid
residues and comprising:
[0016] (1) a first region comprising at least one selected from the
group consisting of an amino acid sequence represented by CSYYQSC
(SEQ ID NO:1) and an amino acid sequence represented by RGD;
and
[0017] (2) a second region comprising [0018] (2-i) an amino acid
sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID
NO:2), [0019] (2-ii) an amino acid sequence having an identity of
not less than 50% to an amino acid sequence represented by SEQ ID
NO:2 and having an adsorption ability to a cultivation container,
or [0020] (2-iii) an amino acid sequence that is the amino acid
sequence represented by SEQ ID NO:2, in which from 1 to 30 amino
acid residues are added, substituted, or deleted, and that has an
adsorption ability to a cultivation container.
[0021] [2] The polypeptide according to [1], wherein a GRAVY value
is from -2.0 to -0.95.
[0022] [3] The polypeptide according to [1] or [2], wherein the
first region comprises both of the amino acid sequence represented
by CSYYQSC (SEQ ID NO:1) and the amino acid sequence represented by
RGD.
[0023] [4] The polypeptide according to any one of [1] to [3],
wherein the polypeptide consists of from 40 to 400 amino acid
residues.
[0024] [5] T The polypeptide according to any one of [1] to [4],
further comprising a third region consisting of any one amino acid
sequence of the following (3-i) to (3-iii):
[0025] (3-i) an amino acid sequence consisting of the 56th to 341st
amino acid residues in an amino acid sequence represented by SEQ ID
NO:3, or a partial amino acid sequence thereof;
[0026] (3-ii) an amino acid sequence having an identity of not less
than 50% to the (3-i) amino acid sequence or partial amino acid
sequence thereof; or
[0027] (3-iii) an amino acid sequence that is the (3-i) amino acid
sequence or partial amino acid sequence thereof in which from 1 to
30 amino acid residues are added, substituted, or deleted.
[0028] [6] The polypeptide according to any one of [1] to [4],
further comprising a third region consisting of any one amino acid
sequence of the following (3a-i) to (3a-iii):
[0029] (3a-i) an amino acid sequence consisting of the 132th to
341st amino acid residues in an amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof;
[0030] (3a-ii) an amino acid sequence having an identity of not
less than 50% to the (3a-i) amino acid sequence or partial amino
acid sequence thereof; or
[0031] (3a-iii) an amino acid sequence that is the (3a-i) amino
acid sequence or partial amino acid sequence thereof in which from
1 to 30 amino acid residues are added, substituted, or deleted.
[0032] [7] The polypeptide according to any one of [1] to [4],
further comprising a third region consisting of any one amino acid
sequence of the following (3b-i) to (3b-iii):
[0033] (3b-i) an amino acid sequence consisting of the 269th to
341st amino acid residues in an amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof;
[0034] (3b-ii) an amino acid sequence having an identity of not
less than 50% to the (3b-i) amino acid sequence or partial amino
acid sequence thereof; or
[0035] (3b-iii) an amino acid sequence that is the (3b-i) amino
acid sequence or partial amino acid sequence thereof in which from
1 to 30 amino acid residues are added, substituted, or deleted.
[0036] [8] The polypeptide according to any one of [1] to [7],
further comprising a fourth region consisting of any one amino acid
sequence of the following (4-i) to (4-iii):
[0037] (4-i) an amino acid sequence consisting of the 374th to
459th amino acid residues in an amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof;
[0038] (4-ii) an amino acid sequence having an identity of not less
than 50% to the (4-i) amino acid sequence or partial amino acid
sequence thereof; or
[0039] (4-iii) an amino acid sequence that is the (4-i) amino acid
sequence or partial amino acid sequence thereof in which from 1 to
30 amino acid residues are added, substituted, or deleted.
[0040] [9] The polypeptide according to any one of [1] to [8],
wherein the (2-ii) amino acid sequence has an identity of not less
than 80% to the amino acid sequence represented by SEQ ID NO:2.
[0041] [10] The polypeptide according to any one of [1] to [8],
wherein the (2-iii) amino acid sequence is the amino acid sequence
represented by SEQ ID NO:2 in which from 1 to 15 amino acid
residues are added, substituted, or deleted.
[0042] [11] A polypeptide consisting of from 80 to 450 amino acid
residues and comprising:
[0043] (1) a first region consisting of an amino acid sequence
consisting of the 25th to 47th amino acid residues in an amino acid
sequence represented by SEQ ID NO:3;
[0044] (2) a second region consisting of an amino acid sequence
consisting of the 342nd to 373rd amino acid residues in the amino
acid sequence represented by SEQ ID NO:3; and
[0045] at least one selected from the group consisting of the
following third and fourth regions:
[0046] (3) the third region consisting of an amino acid sequence
consisting of the 269th to 341st amino acid residues in the amino
acid sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof; and
[0047] (4) the fourth region consisting of an amino acid sequence
consisting of the 374th to 459th amino acid residues in the amino
acid sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof.
[0048] [12] A polypeptide consisting of from 100 to 450 amino acid
residues and comprising:
[0049] (1) a first region consisting of an amino acid sequence
consisting of the 1st to 55th amino acid residues in an amino acid
sequence represented by SEQ ID NO:3;
[0050] (2) a second region consisting of an amino acid sequence
consisting of the 342nd to 373rd amino acid residues in the amino
acid sequence represented by SEQ ID NO:3; and
[0051] at least one selected from the group consisting of the
following third and fourth regions:
[0052] (3) the third region consisting of an amino acid sequence
consisting of the 269th to 341st amino acid residues in the amino
acid sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof; and
[0053] (4) the fourth region consisting of an amino acid sequence
consisting of the 374th to 459th amino acid residues in the amino
acid sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof
[0054] [13] The polypeptide according to [11] or [12], wherein the
polypeptide has a GRAVY value of from -2.0 to -0.95.
[0055] [14] The polypeptide according to any one of [1] to [13],
the number of amino acid residues of which being 250 or less.
[0056] [15] The polypeptide according to any one of [5] to [14],
wherein the polypeptide comprises the third region, and the third
region comprises an amino acid residue other than a cysteine
residue at a position corresponding to a cysteine residue in the
amino acid sequence represented by SEQ ID NO:3.
[0057] [16] The polypeptide according to any one of [5] to [14],
wherein the polypeptide comprises the third region, and the third
region comprises a serine residue, an alanine residue, or a glycine
residue at a position corresponding to a cysteine residue in the
amino acid sequence represented by SEQ ID NO:3.
[0058] [17] The polypeptide according to any one of [1] to [16],
wherein the first region is located at an N-terminal side of the
second region.
[0059] [18] The polypeptide according to any one of [1] to [17],
wherein two cysteine residues in the amino acid sequence
represented by SEQ ID NO:1 are cross-linked to each other.
[0060] [19] A polypeptide comprising an amino acid sequence
represented by any of SEQ ID NO:4 to SEQ ID NO:23, SEQ ID NO:38, or
SEQ ID NO:39.
[0061] [20] A polypeptide, comprising an amino acid sequence
represented by CSYYQSC (SEQ ID NO:1), and consisting of from 40 to
450 amino acid residues that have an adsorption ability to a
cultivation container.
[0062] [21] The polypeptide according to [20], further comprising
an amino acid sequence represented by RGD.
[0063] [22] The polypeptide according to [20] or [21], wherein the
polypeptide has a GRAVY value of from -2.0 to -0.95.
[0064] [23] A method of culturing a pluripotent stem cell,
comprising:
[0065] applying the polypeptide according to any one of [1] to [22]
to a cell culture surface of a support, to obtain a
polypeptide-coated culture surface; and
[0066] seeding a pluripotent stem cell on the polypeptide-coated
culture surface and culturing the pluripotent stem cell.
[0067] [24] The method of culturing a pluripotent stem cell
according to [23], wherein the pluripotent stem cell is at least
one selected from the group consisting of embryonic stem cells,
induced pluripotent stem cells, somatic stem cells, fertilized egg
inner cell mass cells, and early embryonic cells.
[0068] [25] The method of culturing a pluripotent stem cell
according to [23] or [24], wherein the pluripotent stem cell is an
induced pluripotent stem cell.
[0069] [26] The method of culturing a pluripotent stem cell
according to any one of [23] to [25], wherein the pluripotent stem
cell is cultured in the absence of a component derived from a
heterologous animal and a component derived from serum.
[0070] [27] The method of culturing a pluripotent stem cell
according to any one of [23] to [26], wherein an amount of
application of the polypeptide to the cell culture surface is from
1 pmol/cm.sup.2 to 1000 pmol/cm.sup.2.
[0071] [28] A cultivation container comprising: a support
comprising a cell culture surface; and the polypeptide according to
any one of [1] to [22], placed on the cell culture surface of the
support.
Effects of Invention
[0072] According to the invention, a polypeptide that enables a
pluripotent stem cell to proliferate in an undifferentiated state,
does not require treatment of immobilization to a cultivation
container by chemical bonding, and can be industrially produced,
and a method of culturing a pluripotent stem cell using the
polypeptide can be provided.
BRIEF DESCRIPTION OF DRAWINGS
[0073] FIG. 1 is a graph indicating the results of the adsorption
test of each polypeptide to the surface of a culture plate in
Examples of the invention.
[0074] FIG. 2 is a graph indicating the proliferation curve of iPS
cells using each polypeptide in Examples of the invention.
[0075] FIGS. 3 A to 3E represent the morphological images (left
column) and magnified images (right column) of iPS cell colonies in
a case of being cultured on each polypeptide in Examples of the
invention.
[0076] FIGS. 4A to 4E represent the images of DAPI staining (left
column) and NANOG staining (right column) of iPS cells in a case of
being cultured on each polypeptide in Examples of the
invention.
DESCRIPTION OF EMBODIMENTS
[0077] The polypeptide of the invention is: a polypeptide including
(1) an amino acid sequence represented by CSYYQSC (SEQ ID NO:1),
and consisting of from 40 to 450 amino acid residues that have an
adsorption ability to a cultivation container; or a polypeptide
consisting of from 40 to 450 amino acid residues and including: (1)
a first region including at least one selected from the group
consisting of an amino acid sequence represented by CSYYQSC (SEQ ID
NO:1) and an amino acid sequence represented by RGD; and (2) a
second region including (2-i) an amino acid sequence represented by
PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID NO:2), (2-ii) an amino
acid sequence having an identity of not less than 50% to an amino
acid sequence represented by SEQ ID NO:2 and having an adsorption
ability to a cultivation container, or (2-iii) an amino acid
sequence that is the amino acid sequence represented by SEQ ID
NO:2, in which from 1 to 30 amino acid residues are added,
substituted, or deleted, and that has an adsorption ability to a
cultivation container. Hereinafter, the polypeptides may also be
referred to as "polypeptides for use in culture" in the present
specification. Note that the polypeptides may also be used for
purposes other than use in culture.
[0078] It was found in the invention that the polypeptide for use
in culture according to the invention enables cells, particularly
pluripotent stem cells, to favorably proliferate since the first
region containing a specific amino acid sequence has excellent cell
adhesiveness. The polypeptide of the invention having such an amino
acid sequence enables pluripotent stem cells to proliferate in a
long term while maintaining an undifferentiated state.
[0079] It was further found in the invention that the second region
containing a specific sequence contributes to adsorption to a
surface of a cultivation container. The polypeptide of the
invention having such an amino acid sequence exhibits favorable
adhesiveness to a cultivation container, and an incorporation
thereof in combination with the first region into the polypeptide
enables pluripotent stem cells to proliferate in a long term while
maintaining an undifferentiated state without separating from a
cell culture surface of the cultivation container during a culture
period. Further, since the polypeptide of the invention enables
pluripotent stem cells in an undifferentiated state on culture to
proliferate while being suppressed from separating from the surface
of the cultivation container, and can improve handleability in a
culture manipulation.
[0080] As a result, according to the invention, the polypeptide
that promotes proliferation of pluripotent stem cells in an
undifferentiated state, does not require treatment of
immobilization to a cultivation container by chemical bonding, and
can be industrially produced can be obtained.
[0081] The polypeptide according to the invention can eliminate the
risk of contamination of an antigenic substance and an infection
source compared to natural human vitronectin, and can retain
features equivalent to those of natural vitronectin, i.e.,
adhesiveness to a pluripotent stem cell, cell proliferation
properties, and undifferentiation maintenance properties.
[0082] The possibility of contamination of pluripotent stem cells
cultured in a presence of the polypeptide according to the
invention (preferably, further in the absence of a component
derived from a heterologous animal or the like) with a foreign
substance such as an antigenic substance derived from a sample or
the like can be almost fully, or greatly eliminated. Safety of the
pluripotent stem cells cultured in the culturing method can be
sufficiently secured for use in medical applications or
applications equivalent thereto.
[0083] The culturing method using the polypeptide of the invention
enables pluripotent stem cells to be cultured at a lower cost and
in a simple manipulation, and can widely contribute to demands not
only in medical applications but also in research fields.
[0084] The term "step" as used herein encompasses not only an
independent step but also a step that cannot be clearly
distinguished from other steps, as long as the predetermined action
of this step is achieved thereby.
[0085] In addition, a numerical value range expressed by "from (a
minimum value) to (a maximum value)" represents a range which
includes the values shown before and after the "to" as the minimum
and maximum values, respectively.
[0086] In the case of mentioning of an amount of a certain
component in a composition, in a case in which plural substances
corresponding to the component are present in a composition, the
amount means the total amount of the plural substances present in
the composition, unless otherwise specified.
[0087] As used herein, the term "homologous" means a human while
the term "heterologous" means an animal other than humans.
[0088] In the present specification, an amino acid residue in an
amino acid sequence may be described in one character (for example,
a glycine residue in "G") or three characters (for example, a
glycine residue in "Gly"), well known in the art.
[0089] Unless otherwise specified, in the invention, "%" for amino
acid sequences in polypeptides is based on the number of amino acid
(or imino acid) residues.
[0090] In a case in which two or more amino acid sequences to be
compared are aligned (alignment) in a manner well known in the art
in a manner that the same amino acid residues are most in
consideration of insertion, deletion, and substitution, the
expression "corresponding amino acid residue" or the like used for
a particular amino acid residue in one amino acid sequence, as used
herein, means an amino acid residue in the other amino acid
sequences, which matches with the position of the particular amino
acid residue in the one amino acid sequence as a reference.
[0091] As used herein, "identity" related to an amino acid sequence
can refer to a value calculated using the BLAST package (see
Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th
Ed--Chapter 18). For example, an identity of not less than 50% to
SEQ ID NO:2 refers to a value of Max. Identities in BLAST of 50 or
more.
[0092] As used herein, vitronectin means human vitronectin, and
specifically, is a polypeptide composed of 495 amino acid residues
in full length, shown in SEQ ID NO:3 described below. It has also
been confirmed that natural vitronectin is a glycoprotein having a
sugar chain in a part of the sequence thereof.
TABLE-US-00001 SEQ ID NO: 3:
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRG
DVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPE
QTPVLKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCS
GKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIE
GPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNISDGFDG
IPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEG
SSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHG
VPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSR
GRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPI
QSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
[0093] <Polypeptide>
[0094] The polypeptide according to the invention (polypeptide for
use in culture) is: a polypeptide including an amino acid sequence
represented by CSYYQSC (SEQ ID NO:1), and consisting of from 40 to
450 amino acid residues that have an adsorption ability to a
cultivation container; or a polypeptide including the following
first and second regions and consisting of from 40 to 450 amino
acid residues:
[0095] (1) a first region including at least one selected from the
group consisting of an amino acid sequence represented by CSYYQSC
(SEQ ID NO:1) and an amino acid sequence represented by RGD
(hereinafter simply referred to as an RGD sequence); and
[0096] (2) a second region including (2-i) an amino acid sequence
represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID NO:2),
(2-ii) an amino acid sequence having an identity of not less than
50% to an amino acid sequence represented by SEQ ID NO:2 and having
an adsorption ability to a cultivation container, or (2-iii) an
amino acid sequence that is the amino acid sequence represented by
SEQ ID NO:2, in which from 1 to 30 amino acid residues are added,
substituted, or deleted, and that has an adsorption ability to a
cultivation container.
[0097] The first region contains at least one selected from the
group consisting of an amino acid sequence represented by SEQ ID
NO:1 and an RGD sequence.
[0098] The amino acid sequence represented by SEQ ID NO:1
corresponds to from 25th to 31th seven amino acid residues in the
amino acid sequence of vitronectin. The RGD sequence is a
cell-adhesive motif corresponding to from 45th to 47th three amino
acid residues in the amino acid sequence of vitronectin. Each of
the amino acid sequences is a sequence located in a site relatively
closer to the N-terminal in natural vitronectin, exhibits
adhesiveness to an undifferentiated pluripotent stem cell, and is
thus presumed to enable pluripotent stem cells maintained in an
undifferentiated state to proliferate. Therefore, polypeptides that
contain none of the amino acid sequences are poor in cell
adhesiveness and are incapable of offering the advantages of the
invention. However, the invention is not restricted by this
theory.
[0099] Two cysteine residues in the amino acid sequence represented
by SEQ ID NO:1 may be cross-linked with each other. As a result, a
higher-order structure, which improves adhesiveness to pluripotent
stem cells, tends to be formed in the amino acid sequence
represented by SEQ ID NO:1.
[0100] As used herein, the expression "enable pluripotent stem
cells to proliferate in undifferentiated state" means that
pluripotent stem cells maintain differentiation potency in a
culture period. Whether or not pluripotent stem cells are in an
undifferentiated state can be determined by a known evaluation
method. For example, the determination is performed by a method
known to those skilled in the art, such as expression of a
molecular marker (e.g., assay of expression by flow cytometry for
SSEA-4, Oct-4, and/or the like, or immunostaining for Oct-4, NANOG,
and/or the like), confirmation of pluripotent differentiation by an
in vitro experiment, or confirmation of teratocarcinogenesis due to
transplantation into immunodeficient mice and the like. Whether or
not the proliferation is caused may be determined by visual
observation using various microscopes by usual methods, a response
test for ALP activity or the like, a technique utilizing flow
cytometry or the like, or another technique. A culture period in
which the differentiation potency of pluripotent stem cells in the
invention is maintained varies according to culture conditions and
the cell state of the pluripotent stem cells, and may be allowed to
be a culture period of, e.g., 1 month.
[0101] It is sufficient that the first region in the polypeptide
for use in culture contains any one selected from the group
consisting of the amino acid sequence represented by SEQ ID NO:1
and the RGD sequence. From the viewpoint of cell adhesiveness and
cell proliferation properties, it is preferable that the first
region in the polypeptide for use in culture contains both of the
sequences.
[0102] The first region may contain an amino acid sequence which is
different from the amino acid sequence represented by SEQ ID NO:1
and the RGD sequence. Examples of such different amino acid
sequences include (1a) an amino acid sequence consisting of the 1st
to 24th amino acid residues, (1b) an amino acid sequence consisting
of the 48th to 55th amino acid residues, (1c) an amino acid
sequence consisting of the 32th to 44th amino acid residues, in the
amino acid sequence of human vitronectin, shown in SEQ ID NO:3, and
combinations thereof, from the viewpoint of the cell adhesiveness
and cell proliferation properties of the first region. Each of the
amino acid sequences from (1a) to (1c) may have a sequence in which
from 1 to 30 amino acid residues are substituted, deleted, or
eliminated so that the cell adhesiveness and cell proliferation
properties of the first region are not deteriorated, and may have
an amino acid sequence having an identity of not less than 50% to
each sequence of the amino acid sequences from (1a) to (1c).
[0103] The first region can contain, in addition to the amino acid
sequence represented by SEQ ID NO:1 and the RGD sequence, at least
one selected from the group consisting of the amino acid sequences
of from (1a) to (1c), and preferably contains both of the amino
acid sequence represented by SEQ ID NO:1 and the RGD sequence, and
an amino acid sequence consisting of the 1st to 55th amino acid
residues in the amino acid sequence represented by SEQ ID NO:3, or
an amino acid sequence that is similar thereto or part thereof,
from the viewpoint of cell adhesiveness and cell proliferation
properties.
[0104] The number of amino acid residues in the first region, the
number of the amino acid residues can be from 3 to 60 from the
viewpoint of cell adhesiveness and proliferation properties, and
preferably from 10 to 55.
[0105] The second region contains the amino acid sequence
represented by SEQ ID NO:2 and consisting of 32 amino acid
residues, and preferably consists of the amino acid sequence
represented by SEQ ID NO:2 from the viewpoint of the easiness of
purification of the polypeptide for use in culture. The amino acid
sequence represented by SEQ ID NO:2 is contained in a part of
hemopexin-like domain II located in a site closer to the C-terminal
of natural vitronectin, and corresponds to a heparin binding domain
including from 342nd to 373rd amino acid residues in the amino acid
sequence represented by SEQ ID NO:3. Hereinafter, the amino acid
sequence represented by SEQ ID NO:2 may be referred to as a heparin
binding domain.
[0106] It is presumed that the polypeptide for use in culture has
an adsorption ability to a cultivation container because of having
the heparin binding domain. As a result, undifferentiated
pluripotent stem cells can be cultured for a long term while
maintaining an undifferentiated state. However, the invention is
not restricted by this theory.
[0107] The polypeptide for use in culture tends to assure the
hydrophilicity of the polypeptide for use in culture, to suppress
the hydrophobic aggregation of the polypeptide, because of
containing the heparin binding domain. As a result, the
purification of the polypeptide for use in culture is facilitated,
and production efficiency can be enhanced.
[0108] As used herein, the expression "having adsorption ability to
cultivation container" means that the amino acid sequence can be
physically adsorbed to a cell culture surface (hereinafter also
simply referred to as a "culture surface") of a cultivation
container of interest without chemical reaction. Whether or not to
have an adsorption ability to a culture surface of a cultivation
container can be evaluated by, for example, determining whether or
not 10 pmol/cm.sup.2 or more of the polypeptide remaining on a
surface of a polystyrene cultivation container subjected to plasma
treatment is present in a case in which a solution containing the
polypeptide is added to the culture plate so that the polypeptide
is 200 pmol/cm.sup.2, left to stand at 37.degree. C. for 2 hours,
and then washed twice with a phosphate buffer.
[0109] The amount of the polypeptide remaining on the surface of
the culture plate can be assayed by an ELISA (Enzyme-Linked
Immunosorbent Assay) method in which the conjugation amount of
antibody that recognizes the polypeptide is quantitated, or by
hydrolyzing the adsorbed polypeptide and quantitating generated
amino acids by HPLC or the like.
[0110] The heparin binding domain may be an amino acid sequence
having an identity of not less than 50%, preferably not less than
80%, more preferably not less than 90%, still more preferably not
less than 95% to the amino acid sequence represented by SEQ ID
NO:2, enabling pluripotent stem cells to proliferate in an
undifferentiated state, and having an adsorption ability to a
cultivation container.
[0111] In addition, the heparin binding domain may comprise an
amino acid sequence that is the amino acid sequence represented by
SEQ ID NO:2 in which from 1 to 30, preferably from 1 to 15, and
preferably from 1 to 6 amino acids are deleted, substituted, or
added, and may be an amino acid sequence having an adsorption
ability to a cultivation container.
[0112] It is sufficient for the polypeptide to have the first
region and the second region. The relative positions of the regions
are not particularly limited. In the polypeptide for use in
culture, the first region is preferably located at an N-terminal
side of the second region.
[0113] The polypeptide for use in culture consists of from 40 to
450 amino acid residues. In the case of less than 40 amino acid
residues, cell adhesiveness, a cell proliferation property, or an
adsorption ability to a cultivation container may be insufficient.
In the case of more than 450 amino acid residues, cell adhesiveness
or a cell proliferation property, and an adsorption ability to a
cultivation container may be prevented from being suitably exerted,
and association, crosslinking, or aggregation between proteins is
facilitated. The polypeptide for use in culture preferably includes
80 or more, more preferably 90 or more, still more preferably 100
or more, and preferably 400 or less, more preferably 250 or less,
still more preferably 170 or less, even more preferably 150 or less
amino acid residues from the viewpoint of inhibiting the
aggregation or the like. Any of the upper or lower limits may be
combined. For example, the polypeptide preferably consists of from
40 to 400 amino acid residues, more preferably consists of from 80
to 250 amino acid residues, still more preferably consists of from
80 to 150 amino acid residues, and even more preferably consists of
from 100 to 150 amino acid residues.
[0114] It is preferable that the polypeptide for use in culture has
a GRAVY value of from -2.0 to -0.95 from the viewpoint of
preventing hydrophobic aggregation. A GRAVY value (Kyte J.,
Doolittle R. F. (1982), J. Mol. Biol, 157: 105-132) represents a
gross mean hydrophobicity of a polypeptide. A higher GRAVY value
means higher hydrophobicity. A GRAVY value of -0.95 or less results
in a tendency to be capable of easily suppressing occurrence of
hydrophobic aggregation. In contrast, a GRAVY value of -2.0 or more
facilitates adsorption to a surface of a cultivation container and
proliferation of undifferentiated cells. The adsorptivity and cell
proliferation properties tend to be improved with increasing the
GRAVY value. The GRAVY value of the polypeptide is more preferably
from -1.70 to -0.975, and still more preferably from -1.60 to
-1.10, in view of compatibility between the suppression of
aggregation and adsorptivity or cell proliferation properties.
Since more aggregation tends to occur with decreasing the number of
amino acid residues, the GRAVY value is preferably from -1.70 to
-0.975, and still more preferably from -1.60 to -1.10, in view of
compatibility between the suppression of aggregation and
adsorptivity or cell proliferation properties, in a case in which
the polypeptide comprises from 80 to 170 amino acid residues.
[0115] A GRAVY value can be adjusted, for example, by increasing or
decreasing the rate of hydrophobic amino acids (e.g., Trp, Tyr,
Phe, Leu, Ile, Val, or Met), or by increasing or decreasing the
number of amino acid residues, in a sequence.
[0116] It is preferable that the polypeptide for use in culture
further has an amino acid sequence which is different from the
first region and the second region. It is preferable that the
polypeptide for use in culture includes a polypeptide shown in SEQ
ID NO:3, i.e., a partial sequence in the amino acid sequence of
human vitronectin, from the viewpoint of suitably exerting cell
adhesiveness and an adsorption ability to a cultivation container.
As a result, the polypeptide for use in culture can acquire
properties similar to those of human vitronectin, for example,
excellent adhesiveness to pluripotent stem cells, and proliferation
properties.
[0117] From the viewpoint of the cell adhesiveness and cell
proliferation properties of the polypeptide for use in culture, an
adsorption ability to a cultivation container, or the suppression
of aggregation, the partial amino acid sequence in human
vitronectin that can be contained in the polypeptide for use in
culture preferably includes at least one selected from the group
consisting of the following third and fourth regions:
[0118] (3) a third region consisting of an amino acid sequence
selected from an amino acid sequence consisting of the 56th to
341st341st amino acid residues in the amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; and
[0119] (4) a fourth region consisting of an amino acid sequence
selected from an amino acid sequence consisting of the 374th to
459th amino acid residues in the amino acid sequence represented by
SEQ ID NO:3, or a partial amino acid sequence thereof.
[0120] For the third region, (3a) an amino acid sequence consisting
of the 132th to 341st amino acid residues in the amino acid
sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof, (3b) an amino acid sequence consisting of the
269th to 341st amino acid residues, or a partial amino acid
sequence thereof, (3c) an amino acid sequence consisting of the
274th to 341st amino acid residues, or a partial amino acid
sequence thereof can be selected, or (3d) an amino acid sequence
consisting of the 294th to 341st amino acid residues, or a partial
amino acid sequence thereof is acceptable, from the viewpoint of a
tendency to suppress hydrophobic aggregation in a case in which the
polypeptide is produced. Hydrophobic aggregation tends to be able
to be reduced by reducing the number of amino acid residues in the
amino acid sequences from (3a) to (3d). Especially, the selection
of the amino acid sequence (3d) is preferred since hydrophobic
aggregation tends to be able to be more reliably suppressed.
[0121] The fourth region can be an amino acid sequence consisting
of the 374th to 459th amino acid residues, or a partial amino acid
sequence thereof, an amino acid sequence consisting of the 374th to
409th amino acid residues, or a partial amino acid sequence
thereof, or an amino acid sequence consisting of the 374th to 379th
amino acid residues, or a partial amino acid sequence thereof, from
the viewpoint of adsorptivity to a culture plate.
[0122] Especially, from the 374th to 379th amino acid residues are
preferred in view of adsorptivity to a culture plate as well as
easiness of suppressing hydrophobic aggregation in a case in which
the polypeptide is produced, and hydrophobic aggregation tends to
be reduced by reducing the number of selected amino acids.
[0123] Each of the partial amino acid sequences in the amino acid
sequences included in the third and fourth regions means an amino
acid sequence including three or more consecutive amino acid
residues in amino acid residues in the specific range. The number
of the amino acid residues in each of the partial amino acid
sequences may be selected so as not to be more than the total
number of the amino acid residues of the polypeptide for use in
culture mentioned above.
[0124] The amino acid sequence and partial amino acid sequence
thereof included in each of the third and fourth regions may be an
amino acid sequence or partial amino acid sequence thereof having a
identity of preferably not less than 80%, more preferably not less
than 90%, more preferably not less than 95%, still more preferably
not less than 95% to the respective amino acid sequence or partial
sequence thereof. These amino acid sequences can be selected so
that the cell adhesiveness and adsorptivity to a cultivation
container of the polypeptide for use in culture are not
deteriorated.
[0125] The amino acid sequence and partial amino acid sequence
thereof, included in each of the third and fourth regions, may be
an amino acid sequence that is the respective amino acid sequence
or partial sequence thereof in which from 1 to 30, preferably from
1 to 15, more preferably from 1 to 5 amino acid residues are
deleted, substituted, or added. The amino acid sequence in which
the amino acid residues are subjected to deletion or the like can
be selected so that the cell adhesiveness and adsorptivity to a
cultivation container, of the polypeptide for use in culture, are
not deteriorated.
[0126] The polypeptide for use in culture tends to have an
advantage of enhancing adsorptivity to a culture plate because the
polypeptide for use in culture contains the third region. The
polypeptide for use in culture tends to have an advantage of
further enhancing adsorptivity to a culture plate because of
containing the fourth region. It is sufficient for the polypeptide
for use in culture to contain either third or fourth region.
[0127] The GRAVY value of the polypeptide for use in culture is
preferably adjusted, for example, by increasing or decreasing the
number of amino acid residues in the amino acid sequences included
in the third and fourth regions, or by substituting deleting, or
adding amino acid residues, and it is more preferable to
particularly adjust the length of the amino acid sequence included
in the third region, from the viewpoint of the easiness of
adjustment.
[0128] The polypeptide for use in culture does not necessarily
contain from 56th to 131th amino acid residues, from 56th to 268th
amino acid residues, from the 269th to 273th amino acid residues,
and/or from the 50th to 293th amino acid residues, in the amino
acid sequence represented by SEQ ID NO:3. An amino acid sequence
consisting of the amino acid residues is presumed not to contribute
to the feature of the polypeptide for use in culture for the
culture of pluripotent cells, and a suitable sequence is selected
from the viewpoint of adsorption to a culture plate.
[0129] In a case in which the third region contains an amino acid
residue corresponding to a cysteine residue in the sequence
represented by SEQ ID NO:3, an amino acid residue different from a
cysteine residue may be located at the position of the cysteine
residue. As a result, intramolecular or intermolecular crosslinking
due to the cysteine residue can be preferably prevented from being
formed. Examples of the different amino acid residue for
substituting the cysteine residue are not particularly limited but
include a serine residue, an alanine residue, and a glycine
residue. Especially, a serine residue or an alanine residue is
preferred in view of having a structure similar to that of
cysteine.
[0130] The polypeptide for use in culture may also comprise
arbitrary additional amino acid residues other than the above as
long as cell adhesiveness and adsorptivity to a cultivation
container are not deteriorated thereby. Examples of sequences
consisting of such other arbitrary amino acid residues include
additional sequences added for easily producing the polypeptide for
use in culture by a recombinant technology. Examples of such
additional sequences include N-terminal methionine residues,
N-terminal GPLG sequences, tag sequences (for example, GST
(glutathione S-transferase), FLAG tag, His tag, and the like), and
linker sequences that can be added between regions (for example,
GGGS, GGGGS, GGGGGS, and the like).
[0131] The polypeptide for use in culture can be produced by an
amino acid synthesis technology or gene-recombination technology
known to those skilled in the art.
[0132] In a case in which the polypeptide for use in culture of the
invention is obtained by the gene-recombination technology,
specifically, first, genes encoding an amino acid sequence of
interest are acquired and incorporated into an expression vector to
produce a recombinant expression vector, which is introduced into
an appropriate host to produce a transformant. The obtained
transformant is cultured in an appropriate culture medium, to
thereby produce a polypeptide of interest, and the polypeptide of
interest is therefore collected from the culture by a usual method,
whereby the polypeptide according to the invention can be
obtained.
[0133] It is preferable that the polypeptide for use in culture is
a polypeptide (A) consisting of from 80 to 450 amino acid residues,
in which the polypeptide (A) includes: (1) the first region
consisting of an amino acid sequence consisting of the 25th to 47th
amino acid residues in the amino acid sequence represented by SEQ
ID NO:3; (2) the second region consisting of an amino acid sequence
consisting of the 342nd to 373rd amino acid residues in the amino
acid sequence represented by SEQ ID NO:3; and at least one selected
from the group consisting of the following third and fourth
regions: (3) the third region consisting of an amino acid sequence
consisting of the 269th to 341st amino acid residues in the amino
acid sequence represented by SEQ ID NO:3, or a partial amino acid
sequence thereof; and (4) the fourth region consisting of an amino
acid sequence consisting of the 374th to 459th amino acid residues
in the amino acid sequence represented by SEQ ID NO:3, or a partial
amino acid sequence thereof, from the viewpoint of cell
proliferation properties, the proliferation potency of
undifferentiated pluripotent stem cells in an undifferentiated
state, and the like.
[0134] It is preferable that the polypeptide for use in culture is
a polypeptide (B) consisting of the 100 to 450 amino acid residues,
in which the polypeptide (B) comprises: (1) the first region
consisting of an amino acid sequence consisting of the 1st to 55th
amino acid residues in the amino acid sequence represented by SEQ
ID NO:3 (containing the amino acid sequence represented by SEQ ID
NO:1 and the RGD sequence); (2) the second region (heparin binding
domain) consisting of an amino acid sequence consisting of the
342nd to 373rd amino acid residues in the amino acid sequence
represented by SEQ ID NO:3; and at least one selected from the
group consisting of the following third and fourth regions: (3) the
third region consisting of an amino acid sequence consisting of the
269th to 341st amino acid residues in the amino acid sequence
represented by SEQ ID NO:3, or a partial amino acid sequence
thereof; and (4) the fourth region consisting of an amino acid
sequence consisting of the 374th to 459th amino acid residues in
the amino acid sequence represented by SEQ ID NO:3, or a partial
amino acid sequence thereof, from the viewpoint of cell
proliferation properties, the proliferation potency of
undifferentiated pluripotent stem cells in an undifferentiated
state, and the like.
[0135] It is preferable that the polypeptide (A) or (B) is a
polypeptide further having a GRAVY value of from -2.0 to -0.95.
[0136] It is preferable that the polypeptide (A) consists of from
80 to 250 amino acid residues.
[0137] It is preferable that the polypeptide (A) is a polypeptide
further having a GRAVY value of from -2.0 to -0.95 and consisting
of from 80 to 250 amino acid residues.
[0138] It is preferable that the polypeptide (A) is a polypeptide
further having a GRAVY value of from -1.70 to -0.975 and consisting
of from 80 to 250 amino acid residues.
[0139] It is preferable that the polypeptide (A) or (B) consists of
from 100 to 250 amino acid residues.
[0140] It is preferable that the polypeptide (A) or (B) is a
polypeptide further having a GRAVY value of from -2.0 to -0.95 and
consisting of from 100 to 250 amino acid residues.
[0141] It is preferable that the polypeptide (A) or (B) is a
polypeptide further having a GRAVY value of from -1.70 to -0.975
and consisting of from 100 to 250 amino acid residues.
[0142] It is preferable that the polypeptide (A) or (B) is a
polypeptide further having a GRAVY value of from -1.70 to -0.975
and consisting of from 100 to 170 amino acid residues.
[0143] Examples of the polypeptide for use in culture are given
below. However, the invention is not limited thereto.
TABLE-US-00002 TABLE 1 SEQ ID Amino Acid Sequence No.
DQESCKGRCTEGFNVDKKCQCDELGSYYQSCCTDYTAECKPQVTRGDVFTMPEDEPSQEECEGS 4
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMA
PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDW
LVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEPRPSLAKK 5
QRFRHRNRKGYRSQRGHSRGRNQN
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEGVPGQVDA 6
AMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEQPQFISRD 7
WHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEF 8
WGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRK
GYRSQRGHSRGRNQN
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 9
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGM
APRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRM
DWLVPATSEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYVVLGSPAPGHL
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 10
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGM
APRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN
DQESCKGRCTEGFNVDKKCQCDELGSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 11
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGM
APRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSR
DQESGKGRCTEGFNVDKKCQCDELGSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 12
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGM
APRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSE
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 13
SLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGM
APRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYD
DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDESQEESEGS 38
EDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNR
KGYRSQRGHSRGRNQN
[0144] <Method of Culturing Pluripotent Stem Cells>
[0145] The method of culturing a pluripotent stem cell of the
invention includes: applying the polypeptide for use in culture to
a cell culture surface of a support, to obtain a culture surface
coated with the polypeptide for use in culture (hereinafter
referred to as a culture surface preparation step); and seeding a
pluripotent stem cell on the culture surface coated with the
polypeptide for use in culture and culturing the pluripotent stem
cell (hereinafter referred to as a culture step).
[0146] In the culture method of the invention, the polypeptide for
use in culture, which has the first domain which enables
pluripotent stem cells to be retained in an undifferentiated state
and the second domain which enables favorable adsorption to a
cultivation container, is adsorbed to the culture surface, and
pluripotent stem cells are seeded and cultured thereon. Therefore,
the pluripotent stem cells can be cultured with maintaining an
undifferentiated state while suppressing removal of the pluripotent
stem cells due to separation thereof from the cell culture
surface.
[0147] The pluripotent stem cells that can be proliferated in a
retained undifferentiated state by culturing the pluripotent stem
cells on the polypeptide for use in culture according to the
invention are the pluripotent stem cells of primate animals, and
specifically encompasses embryonic stem cells (ES cells), induced
pluripotent stem cells (iPS cells), somatic stem cells, fertilized
egg inner cell mass cells, early embryonic cells, and the like. One
kind of the cells may be used, or two or more kinds of the cells
may be used in admixture, if necessary. The iPS cells encompass
cells described in Nature, 2007, July 19; Vol. 448, pp. 313-317;
Cell, 2006, August 25; Vol. 126(4), pp. 663-676, or cells similar
thereto.
[0148] Especially, examples of pluripotent stem cells that are
preferably applied in the invention include iPS cells.
[0149] Examples of primate animals include human, monkey, and
gorilla, and human congeneric to the polypeptide for use in culture
is particularly preferred. The component or substance applied to
the invention can be preferably applied as a component or substance
derived from a homologous animal to the invention in a case in
which the component or substance applied to the invention is a
component or substance derived from a primate animal.
[0150] A culture liquid used for culture can be appropriately
selected depending on the kind of a cell to be cultured. The
culture liquid that can be used may be any known culture liquid,
and examples thereof include DMEM, MEM, F12, DME, RPMI1640,
MCDB104, 199, MCDB153, L15, SkBM, and Basal medium. To these
culture liquids, various components that can be generally added,
such as glucose, FBS (fetal bovine serum) or human serum, and
antibiotics (such as penicillin and streptomycin) may be added. A
concentration of serum in a case of adding the serum can be
appropriately varied, according to a culture state in the case, and
can be typically set to 10% (v/v).
[0151] It is preferable to culture the pluripotent stem cells in
the absence of a component derived from a heterologous animal in
the culture method. Thereby, the possibility of a contamination of
a foreign substance derived from a heterologous animal can be
eliminated with high precision. Examples of the culture in the
absence of a component derived from a heterologous cell include a
culture in which a culture liquid that does not contain any
component derived from a heterologous animal is used, and a culture
in which feeder cells and the like derived from a heterologous
animal are not used.
[0152] It is preferable to culture the pluripotent stem cells in
the absence of a component derived from a heterologous animal and a
serum component in the culture method. Thereby, a contamination of
a component derived from a heterologous animal can be still more
eliminated.
[0153] As the culture liquid that does not contain any component
derived from a heterologous animal, a mixed culture medium
including a hyposmotic culture medium containing at least one of
medium components such as non-essential amino acids, glutamic acid,
.beta.-mercaptoethanol, FGF-2, TGF-.beta., insulin, and transferrin
can be used. Specifically, a culture medium such as TeSR2 (StemCell
Technologies, Inc.) can be used. However, the culture liquid is not
limited thereto.
[0154] A culture in an incubator under usual culture conditions,
for example, at a temperature of 37.degree. C. and a CO.sub.2
concentration of 5% (v/v) is applied to the cell culture.
[0155] Usual culture media used for maintaining pluripotent stem
cells can be used in methods of culturing and subculturing the
pluripotent stem cells. Specific examples thereof include mTeSR and
TeSR2 (StemCell Technologies, Inc.). The pluripotent stem cells are
seeded in a culture medium by a usual method. The culture media
used in a series of subcultures are not necessarily the same, and
may be different culture media as long as the pluripotent stem
cells can be maintained in an undifferentiated state.
[0156] The culture surface preparation step includes applying a
coating solution containing a predetermined amount of the
polypeptide for use in culture to a culture surface of a support.
As a result, the culture surface can be coated with the polypeptide
for use in culture. A content of the polypeptide for use in culture
in the coating solution varies according to the kind or size of the
culture surface to be coated, and is preferably from 1
pmol/cm.sup.2 to 1000 pmol/cm.sup.2, and more preferably from 100
pmol/cm.sup.2 to 300 pmol/cm.sup.2, from the viewpoint of an
adsorption ability to the culture surface. An aqueous medium used
for preparing the coating solution is not particularly limited, and
examples thereof include phosphate buffers, Tris buffers, and
ultrapure water.
[0157] It is sufficient that the coating solution is applied, and
the coating is thereafter retained for predetermined time, for
example, around from 30 minutes to 24 hours. Thereby, the culture
surface can be coated with the polypeptide for use in culture
without requiring a special treatment.
[0158] The culture step includes seeding the pluripotent stem cells
on the culture surface coated with the polypeptide for use in
culture, and culturing the pluripotent stem cells.
[0159] The seeding density and culture of the pluripotent stem
cells are not particularly limited, and generally used conditions
may be applied. The culture may be carried out under the culture
and subculture conditions mentioned above, for example, at a
seeding density of from around 1.times.10.sup.3/cm.sup.2 to
1.times.10.sup.5/cm.sup.2. A cell mass of from 10 .mu.m to 100
.mu.m may also be cultured under the culture and subculture
conditions mentioned above at a seeding density of from around
1/cm.sup.2 to 5/cm.sup.2.
[0160] Thereby, the pluripotent stem cells can be favorably
proliferated on the polypeptide for use in culture, with favorable
handleability and in a maintained undifferentiated state.
[0161] <Cultivation Container>
[0162] In the invention, the cultivation container refers to a
support having a surface to be used for cell culture. As such a
support, a support well known as a support for cell culture in the
art can be used as it is. Examples of the support may include
plastics (for example, polystyrene, acrylonitrile-butadiene-styrene
resins, polycarbonate resins, and polyester resins), glass,
microporous filters (for example, cellulose, nylon, glass fibers,
polyesters, and polycarbonates), materials for bioreactors (which
may include hollow fiber tubes or microcarrier beads) used in cell
culture in a batch-type or continuous-type process, or in genetic
engineering (for example, bioreactors and the like), polyethylene
terephthalate, Teflon (registered trademark), ceramics, and related
polymer materials.
[0163] The support may also be a support of which a culture surface
is coated with a plasma polymerized thin film.
[0164] A form of the cultivation container is not particularly
limited, and may be any form that can be applied to the culture of
the pluripotent stem cells. Examples of containers with such forms
include multi-well plates (for example, 6-well, 12-well, 24-well,
and 96-well), culture plates (for example, petri dishes and the
like), tubes, culture flasks, roller bottles, and shake culture
flasks.
[0165] The cultivation container according to the invention has a
support having a cell culture surface, and the polypeptide for use
in culture, placed on the cell culture surface of the support.
[0166] Since the cultivation container has the culture surface
provided with the polypeptide for use in culture according to the
invention, the polypeptide for use in culture is favorably adsorbed
to the culture surface, and pluripotent stem cells can be
proliferated with favorable handleability and in a maintained
undifferentiated state in a case in which the pluripotent stem
cells are seeded on the polypeptide for use in culture.
[0167] As used herein, the culture surface in the cultivation
container means a surface to which cells can adhere in a case in
which the seeded cells are grown.
[0168] The cultivation container according to the invention can be
produced by a production method including: preparing a material
provided with a support having a cell culture surface (hereinafter
"preparation step"); and applying the polypeptide for use in
culture to the cell culture surface, to perform adsorption
treatment (hereinafter "adsorption treatment step"). As a result,
the cultivation container according to the invention can be easily
obtained.
[0169] In the preparation step, the cultivation container provided
with the support having the culture surface is prepared. In a case
in which the support has a plasma polymerized thin film on the
culture surface, a step of forming the plasma polymerized thin film
on the support may be included therein. To the method of forming
the plasma polymerized thin film, a usual method may be applied
without being changed.
[0170] The adsorption treatment step comprises applying the
polypeptide for use in culture according to the invention to the
culture surface, and retaining the polypeptide for use in culture.
In the adsorption treatment step, an adsorption liquid containing a
predetermined amount of the polypeptide for use in culture may be
prepared, applied to the culture surface, and retained for
predetermined time, to thereby adsorb the polypeptide for use in
culture to the culture surface.
[0171] The explanation of the step of preparing the culture surface
coated with the polypeptide for use in culture in the culture
method can be applied to the adsorption treatment step as it
is.
EXAMPLES
[0172] The invention will be described in detail below by examples.
However, the invention is not limited to these examples. Unless
otherwise specified, "%" is based on mass.
Example 1
Preparation of Polypeptide
[0173] A gene sequence encoding each polypeptide of from RCP-1 to
RCP-17 having amino acid sequences listed in Table 2 and Table 3
was amplified by a usual method utilizing PCR. RCP-11 corresponds
to the sequence of natural human vitronectin. A position
corresponding to the amino acid sequence of each polypeptide in the
amino acid sequence (SEQ ID NO:3) of natural human vitronectin is
listed in each "NOTE" box in Table 2 and Table 3. It is remarked
that the amino acid sequences of the polypeptides may include an
amino acid sequence which is the amino acid sequence of a
corresponding range in natural human vitronectin as described in
each table and to which addition, deletion, or substitution is
performed. Each of from RCP-1 to RCP-10, and RCP-17 has the same
amino acid sequence as each of the amino acid sequences shown in
SEQ ID NOs: 4 to 13 and SEQ ID NO:38 mentioned above except that
methionine is present at position 1.
[0174] For from RCP-1 to RCP-10, and RCP-17, genes of interest were
inserted into pET-28b (+), previously subjected to cleavage
treatment with NcoI (Takara Bio Inc.), using an InFusion Advantage
PCR Cloning Kit (Clontech), to construct each vector for
expression. For from RCP-11 to RCP-16, genes of interest were
inserted into pGEX-6P-1 (GE Healthcare) previously subjected to
cleavage treatment with BamHI (Takara Bio Inc.) by the same
technique as described above, to construct each vector for
expression. The sequence of each vector for expression was
confirmed by sequence analysis.
TABLE-US-00003 TABLE 2 SEQ ID Amino Acid Sequence NO: NOTE The
RCP-1 MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPE 14
1-55 Invention
DEPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDW 269-459
HGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNS
RRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRV
NLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL RCP-2
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP 15 1-55
EDEPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN 342-373 RCP-3
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP 16 1-55
EDEGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRN 322-341 QN
342-373 RCP-4 MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP
17 1-55 EDEQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYR
312-341 SQRGHSRGRNQN 342-373 RCP-5
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP 18 1-55
EDEFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQR 302-341
FRHRNRKGYRSQRGHSRGRNQN 342-373 RCP-6
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP 19 1-55
EDESQEESEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRD 269-459
WHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQ C274S
NSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATSEPIQSVFFFSGDKY
YRVNLRTRRVDTVDPPYPRSIAQYWLGSPAPGHL RCP-7
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPE 20 1-55
DESQEESEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWH 269-373
GVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN C274S RCP-8
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPE 21 1-55
DESQEESEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWH 269-373
GVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSR 374-379 RPSR
C274S RCP-9 MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPE
22 1-55 DESQEESEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWH
269-373 GVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSR
374-389 RPSRATWLSLFSSE C274S RCP-10
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPE 23 1-55
DESQEESEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWH 269-373
GVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSR 374-399
RPSRATWLSLFSSEESNLGANNYD C274S RCP-17
MDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMP 39 1-55
EDESQEESEGSEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIY 269-277
ISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN 295-341 342-373 C274S
TABLE-US-00004 TABLE 3 SEQ ID Amino Acid Sequence NO: NOTE
Comparative RCP-11
GPLGDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDV 24 1-459
Example FTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPE
EEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGS
LFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFK
GSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFK
GKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSA
GTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGY
RSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCE
PIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL RCP-12
GPLGDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGD 25 1-55
VFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLK 56-268
PEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLK
NGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKT
YLFKGSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRER
VYFFKGKQYWEYQFQHQ RCP-13
GPLGDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDV 26 1-55
FTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKP 56-129
EEEAPAPEVGASKPEGIDSRPETLHPGRPQP RCP-14
GPLGDQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGD 27 1-55
VFTMPEDE RCP-15
GPLGYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEEA 28 56-459
PAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFA
FRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQ
YWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQ
YWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTR
QPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRPRHRNRKGYRSQ
RGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQ
SVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL RCP-16
GPLGPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFI 29 269-459
SRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSR
GRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFF
SGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
[0175] Each of the produced expression vectors of from RCP-1 to
RCP-10, and RCP-17 was transformed into BL21 (DE3) pLysS (Novagen)
by a usual method, applied to a kanamycin-containing LB plate, and
incubated at 37.degree. C. for 16 hours. The introduction of each
vector was confirmed by a colony direct PCR method, followed by
adding 1 mM of IPTG (Wako Pure Chemical Industries, Ltd.) and by
culturing the resultant with shaking at 37.degree. C. for 5 hours,
to induce the expression of a polypeptide.
[0176] A microbial body was collected by centrifugal treatment, and
the microbial body was resuspended in a washing buffer (20 mM Tris,
150 mM NaCl, pH 7.6). The microbial body was crushed by sonication
and thereafter centrifuged at 4.degree. C. and 15000 rpm for 30
min, and an insoluble fraction was collected. The insoluble
fraction was washed with a washing buffer containing 0.5 mass % of
Triton X100, thereafter resuspended in a low-concentration urea
buffer (Low Urea Buffer: 20 mM Tris, 150 mM NaCl, 2 M urea, pH
7.6), and subjected to sonication treatment. An insoluble fraction
was collected by centrifugal treatment, followed by adding a high
concentration urea buffer (High Urea Buffer: 20 mM Tris, 150 mM
NaCl, 8 M urea, pH 7.6) thereto and by solubilizing the insoluble
fraction by sonication treatment.
[0177] The solution containing a peptide of interest, obtained by
the above-described method, was purified using an AKTA EXPLORER 100
(GE Healthcare) and a HITRAP HEPARIN HP 5 ml (GE Healthcare).
Stepwise elution was performed with the high concentration urea
buffer as a binding buffer and with a high-salt-concentration
adjustment buffer (20 mM Tris, 1 M NaCl, 8 M urea, pH 7.6) as an
elution buffer, to purify a polypeptide of interest.
[0178] Each of the expression vectors of from RCP-11 to RCP-16
produced as described above was transformed into BL21 (Novagen) by
a usual method, applied to an ampicillin-containing LB plate, and
incubated at 37.degree. C. for 16 hours. The introduction of each
vector was confirmed by a colony direct PCR method, followed by
adding 100 .mu.M of IPTG (isopropyl-.beta.-D-thiogalactopyranoside)
and by culturing the resultant with shaking at 20.degree. C. for 24
hours, to induce the expression of a polypeptide.
[0179] A microbial body was collected and resuspended in B-PER
(registered trademark) BACTERIAL PROTEIN EXTRACTION REAGENT in
Phosphate Buffer (Thermo Scientific Inc.), followed by crushing the
microbial body by sonication. The microbial body was centrifuged at
4.degree. C. and 15000 rpm for 30 min, to remove an insoluble
fraction. A supernatant was purified using an AKTA EXPLORER 100 and
a GSTRAPHP 5 ml.times.2 (GE Healthcare). An elution fraction was
desalted using a HIPREP 26/10 DESALTING (GE Healthcare), a protease
for cleaving a GST fusion protein (PreScission Protease) was
further added in 1/2000 of the amount of solution, and the
resultant was incubated at 4.degree. C. for 24 hours, to cleave a
GST tag. Purification was performed in the GSTRAPHP 5 ml.times.2
again, and the cleaved GST tag was adsorbed to a column, and was
removed. A fraction which passed through the column was dialyzed
using a SLIDE-A-LIZER (3.5K MWCO.: Thermo Fisher Scientific Inc.,
the same applies hereafter), and buffer replacement was performed
with PBS.
[0180] The polypeptide of RCP-1 obtained as described above was
electrophoresed in a ready-made gel (12.5%, Bio-Rad), and was
stained with GELCODE.TM. Blue Stain Reagent (Thermo Scientific
Inc.). As a result, a single band was able to be recognized in a
spot corresponding to a molecular weight of 28.3 kDa, which was
expected from the amino acid sequence. Similar results were
obtained in the other polypeptides.
[0181] For from RCP-1 to RCP-10, and RCP-17, each polypeptide
solution after the purification was dialyzed using a SLIDE-A-LIZER
(3.5K MWCO.). A dialysis external liquid was based on a dialysis
buffer (PBS, 1.5 M NaCl, 0.5 M L-arginine, 1 mM EDTA, pH 7.4), and
urea was removed by stepwise dialysis. The concentration of an end
dialysis product was calculated at an absorbance of 280 nm using a
NANODROP (Thremo Fisher Scientific Inc.). The presence or absence
of aggregation after the dialysis is listed in Table 4.
[0182] A GRAVY value was calculated as a value obtained by dividing
the total sum of hydrophobicity indices of which each is determined
according to each amino acid by the number of amino acids (see Kyte
J., Doolittle R. F. (1982), J. Mol. Biol, 157: 105-132). The GRAVY
value is an index for the hydrophilicity and hydrophobicity of each
polypeptide, calculated from the hydrophobicity of amino acids
contained in each polypeptide, and the higher value exhibits a more
hydrophobic property while the lower value exhibits a more
hydrophilic property. The results are listed in Table 4.
[0183] In addition, the presence or absence of aggregation was
evaluated by G, A, and B below. The results are also listed in
Table 4.
[0184] G: Formation of an aggregate is not observed.
[0185] A: Formation of particles having a particle diameter of
around 100 nm is observed.
[0186] B: Formation of visible aggregation with a particle diameter
of 1 mm or more is observed.
TABLE-US-00005 TABLE 4 The Number of GRAVY Amino Acids Aggregation
RCP-1 -0.835 247 A RCP-2 -1.516 88 G RCP-3 -1.124 108 G RCP-4
-1.150 118 G RCP-5 -1.124 128 G RCP-6 -0.875 246 A RCP-7 -0.979 160
A RCP-8 -1.045 166 A RCP-9 -0.958 176 B RCP-10 -0.971 186 B RCP-17
-1.072 143 A
[0187] As listed in Table 4, it is found that each of from RCP-2 to
RCP-5, from RCP-7 to RCP-8, and RCP-17 causes suppression of
aggregation by having a GRAVY value of from -1.70 to -0.975,
although it is a polypeptide consisting of from 80 to 170 amino
acid residues, which should facilitate aggregation.
Example 2
Evaluation of Adsorptivity to Culture Plate
[0188] Each polypeptide obtained by the above-described method was
diluted with a predetermined buffer so as to be added to wells at a
predetermined final concentration of from 0 to 200 pmol/cm.sup.2,
and the resultant was aliquoted in 64 .mu.L to a 96-well plate made
of polystyrene and treated with plasma (Tissue Culture-Treated,
Falcon). Each polypeptide was adsorbed to the plate by incubation
at 37.degree. C. for 2 hours, and was thereafter washed with PBS
twice, to obtain a surface coated with each of the polypeptides of
from RCP-1 to RCP-16.
[0189] To surfaces coated with RCP-1, and from RCP-11 to RCP-16
among the polypeptide-coated surfaces obtained as described above,
64 .mu.L of each of boric acid buffer and 1 N NaOH was applied, and
incubated at 80.degree. C. and a humidity of 100% for 24 hours.
After air-cooling, 75 .mu.L of boric acid buffer was added to each
well, and 50 .mu.L of reaction liquid in which an OPA
(o-phthalaldehyde: Wako Pure Chemical Industries, Ltd.)/methanol
solution (160 mg/ml) and an NAC (N-acetyl-L-cysteine: Wako Pure
Chemical Industries, Ltd.)/boric acid buffer solution (2 mg/ml)
were mixed at 1:100 (mass ratio) was further added. After
incubation at 40.degree. C. for 30 minutes, the fluorescence
intensity thereof was measured (excitation of 355 nm/fluorescence
of 486 nm) using an ENVISION MULTI-LABEL COUNTER (PerkinElmer
Inc.). A calibration curve was separately made from each
polypeptide solution, to calculate an amount of adsorption. The
results are listed in FIG. 1. In FIG. 1, a black rhombus, a black
tetragon, a black triangle, a black circle, a white rhombus, a
white tetragon, and a white triangle represent RCP-1, RCP-11,
RCP-12, RCP-13, RCP-14, RCP-15, and RCP-16, respectively.
[0190] As indicated in FIG. 1, it is found that favorable
adsorptivity to a plate, equivalent to that of RCP-11 having the
sequence of human vitronectin, is exhibited in a case in which
among the polypeptides used in the test, the polypeptides of RCP-1,
RCP-15, and RCP-16, containing PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN
(SEQ ID NO:2 [from 342nd to 373rd in SEQ ID NO:3]), are used. In
contrast, it is found that the adsorption amount of RCP-13 or
RCP-14 that does not contain PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN, is
as low as 1/4 of that of the polypeptide containing the sequence,
and RCP-13 and RCP-14 are inadequate as an adsorbent to a culture
plate.
Example 3
Cell Adhesiveness Evaluation 1
[0191] The cell adhesiveness of human iPS cells ("Tic": cell
number: No. JCRB1331: given by the National Institute of Biomedical
Innovation [7-6-8 Saito-Asagi, Ibaraki-shi, Osaka 567-0085, Japan])
to the above-described polypeptides was evaluated as described
below.
[0192] EMBRYOMAX (registered trademark) (primary mouse embryo
fibroblasts: hygromycin resistance, mitomycin C-treated, derived
from C57/BL6, third generation of subculture) (EMD Millipore
Corporation) was used as feeder cells for maintaining the human iPS
cells, was cultured for 24 hours using DMEM (Invitrogen) culture
medium containing a 10% (v/v) fetal bovine serum, and was made to
adhere onto a T25 flask (Corning Inc.). A culture medium for human
iPS cells, in which FGF-2 (Sigma-Aldrich) was added to the
compositions in Table 5 so as to have a final concentration of 10
ng/ml, was used.
TABLE-US-00006 TABLE 5 Composition Manufacturer Amount KO-DMEM/F12
Invitrogen 400 ml Non-Essential Amino Acid Solution 4 ml
L-Glutamine 5 ml Knock Out Serum Replacement 100 ml
2-Mercaptoethanol Wako Pure Chemical 55 mM Industries, Ltd. 0.925
ml Total: 500 ml
[0193] The maintenance culture of the iPS cells was performed in a
5% (v/v, the same applies hereafter) CO.sub.2 incubator at
37.degree. C. using the above-described culture medium. The culture
medium was replaced by a fresh culture medium every day except the
next day after the seeding of the iPS cells. Subculture
manipulation was performed by separating cells with DISPASE II
(neutral protease GradeII, Roche) and by dividing the cells to
suitable sizes by pipetting manipulation.
[0194] The human iPS cells cultured as mentioned above were treated
with TRYPLE SELECT (Invitrogen) at 37.degree. C. for 5 minutes, and
were separated into single cells. The cells were collected by
centrifugation at 300 rpm for 2 min, and were suspended in TESR2
(component derived from a heterologous animal, serum component-free
culture medium, StemCell Technologies, Inc.) containing Y-27362
((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxiamide.2HC-
l.H.sub.2O, Rho-binding kinase inhibitor, Wako Pure Chemical
Industries, Ltd.) at a final concentration of 10 .mu.M.
[0195] Samples from 1 to 17 were prepared so as to have addition
concentrations listed in Table 6 with from RCP-1 to RCP-10, and
RCP-17, RCP-11, RCP-15, and RCP-16, and human vitronectin
(extracted from human plasma, BD Biosciences) and recombinant
laminin (rLaminin-5: Oriental Yeast Co., Ltd., and Human
Recombinant Laminin-511: Biolamina) as references, were added to
each well of a 96-well plate, were retained at 37.degree. C. for 2
hours, and were adsorbed. iPS cells were seeded on each obtained
well of the 96-well plate, which were subjected to the peptide
treatment, in a manner to set a cell density of 30000 cells/well.
After culture for 24 hours, non-adherent cells were removed by PBS
washing, and only the adherent cells were immobilized with 4%
paraformaldehyde (Wako Pure Chemical Industries, Ltd.). ALP
activity was calculated with ATTOPHOS (registered trademark) AP
Fluorescent Substrate System (Promega Corporation), and the number
of undifferentiated iPS cells having ALP activity was calculated
from a calibration curve. The results are listed in Table 6. In
Table 6, cell adhesion rates were relative values in a case in
which the cell adhesion rate of the sample 15, in which the natural
vitronectin was used, was regarded as 100. n=3.
TABLE-US-00007 TABLE 6 Cell Adhesion Kind of Peptide Addition
Amount Rate (%) Remarks Sample 1 RCP-1 200 pmol/cm.sup.2 109.3 .+-.
5.3 The Invention Sample 2 RCP-2 20 .mu.g/cm.sup.2 98.7 .+-. 6.2
The Invention Sample 3 RCP-3 20 .mu.g/cm.sup.2 106.1 .+-. 4.5 The
Invention Sample 4 RCP-4 20 .mu.g/cm.sup.2 100.1 .+-. 4.5 The
Invention Sample 5 RCP-5 10 .mu.g/cm.sup.2 94.1 .+-. 6.5 The
Invention Sample 6 RCP-6 20 .mu.g/cm.sup.2 92.8 .+-. 4.4 The
Invention Sample 7 RCP-7 5 .mu.g/cm.sup.2 88.6 .+-. 8.1 The
Invention Sample 8 RCP-8 5 .mu.g/cm.sup.2 89.2 .+-. 1.4 The
Invention Sample 9 RCP-9 20 .mu.g/cm.sup.2 95.5 .+-. 10.2 The
Invention Sample 10 RCP-10 20 .mu.g/cm.sup.2 93.0 .+-. 7.8 The
Invention Sample 11 RCP-17 5 .mu.g/cm.sup.2 95.9 .+-. 4.6 The
Invention Sample 12 RCP-11 200 pmol/cm.sup.2 93.5 .+-. 7.9
Comparative Example Sample 13 RCP-15 200 pmol/cm.sup.2 13.2 .+-.
3.4 Comparative Example Sample 14 RCP-16 200 pmol/cm.sup.2 9.4 .+-.
2.9 Comparative Example Sample 15 Natural vitronectin 130
pmol/cm.sup.2 100 .+-. 5.5 Comparative Example Sample 16 rLaminin-5
3.2 .mu.g/cm.sup.2 155.7 Comparative Example Sample 17 Laminin-511
5.0 .mu.g/cm.sup.2 142.0 Comparative Example
[0196] As listed in Table 6, the cell adhesion rates of iPS cells
of from RCP-1 to RCP-10, RCP-17, and RCP-11, and the natural human
vitronectin, having from 1st to 55th in the sequence represented by
SEQ ID NO:3, were favorable. In particular, the cell adhesion rates
of from RCP-1 to RCP-10, and RCP-17, which do not contain some or
all of from 56th to 268th amino acids of the sequence represented
by SEQ ID NO:3, were better than those of the natural human
vitronectin and RCP-11 having the same amino acid sequence as that
of the natural human vitronectin. This reveals that a sequence
important for cell adhesion is present in from 1st to 55th in the
sequence represented by SEQ ID NO:3.
Example 4
Cell Adhesiveness Evaluation 2
[0197] Polypeptides listed in Table 7 were synthesized by Fmoc
solid phase synthesis. A surface to which natural vitronectin was
adsorbed at a concentration of 130 pmol/cm.sup.2, and thereafter, a
cell suspension to which 100 .mu.M of the above-described synthetic
peptide was added was seeded at a rate of 30,000 cells/well. The
number of adherent cells after 24 h post seeding was calculated by
the same technique as in <Cell Adhesiveness Evaluation 1>.
The results are listed in Table 7. In Table 7, the cell adhesion
rates were relative values in a case in which the cell adhesion
rate in a culture liquid that does not contain any synthetic
peptide was regarded as 100. n=3.
TABLE-US-00008 TABLE 7 Synthetic Peptide Cell Adhesion SEQ ID
Sequence Rate (%) NO: Peptide-1 DQESCKGRCTEGFNVDKKCQ 91.8 .+-. 1.2
30 Peptide-2 KGRCTEGFNVDKKCQCDELC 92.7 .+-. 19.6 31 Peptide-3
EGFNVDKKCQCDELCSYYQS 102.5 .+-. 4.2 32 Peptide-4
DKKCQCDELCSYYQSCCTDY 63.8 .+-. 11.6 33 Peptide-5
CCTDYTAECKPQVTRGDVFT 70.5 .+-. 7.1 34 Peptide-6
TAECKPQVTRGDVFTMPEDE 52.7 .+-. 10.3 35 Peptide-7
CCTDYTAECKPQVTRGEVFT 86.7 .+-. 7.1 36 Peptide-8
TAECKPQVTRGEVFTMPEDE 83.8 .+-. 14.8 37
[0198] As listed in Table 7, it is found that cell adhesion to
natural vitronectin was significantly inhibited by adding
Peptide-4, -5, or -6 containing CSYYQSC or RGD, whereas adhesion
inhibition did not occur in a case in which Peptide-1, -2, or -3,
in which neither CSYYQSC nor RGD is contained, or Peptide-7 or -8,
in which the RGD sequence of Peptide-5 or -6 was substituted with
RGE, was added. Accordingly, it is found that a polypeptide
contains at least one of CSYYQSC or RGD, whereby cell adhesiveness
is exhibited.
Example 5
Proliferation Evaluation
[0199] The iPS cells collected in the same manner as in <Cell
Adhesiveness Evaluation 1> described above were seeded on a
96-well plate, to which RCP-1, RCP-11, and the natural human
vitronectin were adsorbed, at a rate of 250 cells/well, and were
cultured in a 5% CO.sub.2 incubator at 37.degree. C. for 8 days.
The number of adherent cells after each lapse of time was measured
by the same method as in <Cell Adhesiveness Evaluation 1>
described above, to obtain proliferation curves. The proliferation
curves were indicated in FIG. 2. In FIG. 2, a black rhombus
represents an example in which RCP-1 was used, while a black
tetragon represents an example in which RCP-11 was used.
[0200] Similarly, each of samples from 1 to 12 was prepared so as
to have an addition concentration listed in Table 8 with each of
from RCP-1 to RCP-10, and RCP-17, and Human Recombinant Laminin-511
as a reference, was seeded on a 96-well plate, to which each
polypeptide was adsorbed, at a rate of 5000 cells/well in the same
manner as in <Cell Adhesiveness Evaluation 1> described
above, and was cultured in a CO.sub.2 incubator at 37.degree. C.
for 3 days. The number of cells after the 3 days was measured by
the same method as in <Cell Adhesiveness Evaluation 1>
described above. The results are listed in Table 8.
TABLE-US-00009 TABLE 8 The Number (%) of Cells Kind of Peptide
Addition Amount after 3 Days Remarks Sample 1 RCP-1 80
.mu.g/cm.sup.2 100.0 The Invention Sample 2 RCP-2 20 .mu.g/cm.sup.2
81.7 The Invention Sample 3 RCP-3 20 .mu.g/cm.sup.2 109.7 The
Invention Sample 4 RCP-4 20 .mu.g/cm.sup.2 120.7 The Invention
Sample 5 RCP-5 10 .mu.g/cm.sup.2 121.2 The Invention Sample 6 RCP-6
20 .mu.g/cm.sup.2 70.5 The Invention Sample 7 RCP-7 5
.mu.g/cm.sup.2 89.9 The Invention Sample 8 RCP-8 5 .mu.g/cm.sup.2
171.0 The Invention Sample 9 RCP-9 20 .mu.g/cm.sup.2 105.8 The
Invention Sample 10 RCP-10 20 .mu.g/cm.sup.2 101.9 The Invention
Sample 11 RCP-17 5 .mu.g/cm.sup.2 153.5 The Invention Sample 12
Laminin-511 1.28 .mu.g/cm.sup.2 56.8 Comparative Example
[0201] FIG. 2 reveals that RCP-1 exhibited a higher cell
proliferation property than that of RCP-11 having the amino acid
sequence of natural vitronectin. The number of cells of RCP-11 was
smaller than that of RCP-1 by around 1/3 of that of RCP-1 on
culture day 8. It was calculated from the change of each obtained
cell number that doubling time was 46.4.+-.2.1 hours in the case of
using RCP-1, and that was 67.7.+-.2.1 hours in a case of using
RCP-11.
[0202] In addition, Table 8 reveals that all of from RCP-1 to
RCP-10, and RCP-17 have higher cell proliferation rates than that
of laminin, which is an extracellular matrix similarly to
vitronectin. It is found that such a high cell proliferation rate
is also obtained in the similar manner in a case in which the 274th
cysteine residue in SEQ ID NO:3 is substituted with a serine
residue.
[0203] The results of FIG. 2 and Table 8 surprisingly reveal that
from RCP-1 to RCP-10, and RCP-17, which include the sequences
effective for cell proliferation and adsorption to a culture plate
and do not contain any sequence corresponding to any or all of from
the 56th to 268th amino acids of natural human vitronectin, have
higher proliferation potencies than RCP-11 having the sequence
equivalent to that of the human vitronectin, and Laminin-511 as a
comparative example.
[0204] In addition, Table 8 reveals that all of from RCP-1 to
RCP-10, and RCP-17, containing both of the sequence of CSYYQSC and
the RGD sequence, and the sequence of
PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN exhibited high cell proliferation
properties.
Example 6
Cell Adhesiveness Evaluation 3
[0205] Cell adhesiveness was evaluated in the same manner as in
<Cell Adhesiveness Evaluation 1> except that RCP-1 was
adjusted with PBS to have a concentration of from 125 pmol/cm.sup.2
to 1000 pmol/cm.sup.2 to be used. The results are listed in Table
9. In Table 9, cell adhesion rates were relative values in a case
in which a cell adhesion rate to a cultivation container to which
natural vitronectin was adsorbed at a concentration of 130
pmol/cm.sup.2 was regarded as 100. n=3.
TABLE-US-00010 TABLE 9 Peptide Addition Amount Cell Adhesion Rate
(%) Remarks RCP-1 1000 pmol/cm.sup.2 98.5 .+-. 17.2 The Invention
RCP-1 500 pmol/cm.sup.2 108.8 .+-. 23.0 The Invention RCP-1 250
pmol/cm.sup.2 89.2 .+-. 10.6 The Invention RCP-1 125 pmol/cm.sup.2
90.3 .+-. 25.3 The Invention Natural vitronectin 130 pmol/cm.sup.2
100 .+-. 5.5 Comparative Example
[0206] As listed in Table 9, as for the adhesiveness of iPS cells
to RCP-1, a cell adhesion rate equivalent to that of natural
vitronectin was exhibited in a case of adding not less than 125
pmol/cm.sup.2.
Example 7
Evaluation of Maintenance of Undifferentiation
[0207] iPS cells collected in the same manner as in <Cell
Adhesiveness Evaluation 1> described above were suspended in
TESR2. Induced pluripotent stem cells (iPS cells) were seeded on a
6-well plate (Tissue Culture-Treated, Falcon) to which each of the
sample 1, the sample 2, the sample 5, the sample 6, and the sample
7 used in <Cell Adhesiveness Evaluation 1> described above
was adsorbed in the same manner as in <Cell Adhesiveness
Evaluation 1>, and were cultured in a CO.sub.2 incubator at
37.degree. C. The culture medium was replaced by a fresh culture
medium every day except the next day after the seeding. Subculture
was performed by the same method as mentioned above every six days.
The forms of the iPS cells cultured on each sample are shown in
FIG. 3.
[0208] The cells were cultured under these conditions for 1 month,
thereafter immobilized with 4% paraformaldehyde, and made to have
membrane permeability enhanced with 1% Triton-X/PBS. Blocking
treatment was performed with an Image IT Signal Enhancer
(Invitrogen), followed by adding an anti-human NANOG antibody
(AF1997, R&D Systems, Inc), ALEXA FLUOR 555-CONJUGATE rabbit
anti-goat IgG antibody (Invitrogen), and DAPI (Dojindo
Laboratories), by performing labeling, and by photographing the
cells with a fluorescence microscope. These fluorescence microscope
images are shown in FIG. 4.
[0209] In each of FIG. 3 and FIG. 4, "A", "B", "C", "D", and "E"
show iPS cells cultured on RCP-1, iPS cells cultured on RCP-11, iPS
cells cultured on the natural human vitronectin, iPS cells cultured
on rLaminin-5, and iPS cells cultured on rLaminin-511,
respectively. Scale bar: 100 .mu.m. In FIG. 3, overall colony
images are shown in the left column, while magnified images are
shown in the right column; and in FIG. 4, DAPI-stained images are
shown in the left column while anti-NANOG antibody-stained images
are shown in the right column. Scale bars in FIGS. 3 and 4: 200
.mu.m.
[0210] As shown in FIG. 3, the iPS cells cultured on RCP-1, RCP-11,
or natural human vitronectin, which contains the sequence effective
for cell proliferation and adsorption to a culture plate, exhibited
a homogeneous colony and a form characteristic for undifferentiated
cells having a high nucleus occupying rate. As shown in FIG. 4, the
iPS cells cultured on RCP-1, RCP-11, or natural human vitronectin
having a cell proliferation domain and an adsorption domain
strongly expressed NANOG in an overall colony, and it was found
that an undifferentiated state was favorably maintained.
[0211] The evaluation results of the above-described examples from
1 to 7 reveal that the polypeptide containing either of CSYYQSC or
the RGD sequence, and the sequence of
PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN, and consisting of from 40 to 450
amino acid residues was excellent in adsorptivity to a cultivation
container. The results also reveal that under conditions of
cocultivation with iPS cells, such a polypeptide exhibited the cell
adhesiveness of the iPS cell, and the maintenance of an
undifferentiated state which are equivalent to those of RCP-11
having the sequence equivalent to those of the natural vitronectin
and human vitronectin, and the properties of proliferation of the
iPS cells which is superior to that of RCP-11. It is found that all
of from RCP-1 to RCP-10, and RCP-17 are favorable in view of the
cell adhesiveness of iPS cells and the maintenance of an
undifferentiated state. Such favorable results in all the abilities
were not able to be obtained in the other polypeptides or the
recombinant laminin as the comparative example.
[0212] Accordingly, the invention can provide a polypeptide that
enables a pluripotent stem cell to proliferate in an
undifferentiated state and is excellent in adsorptivity to a cell
culture surface, a method of culturing a pluripotent stem cell
using the polypeptide, and a cultivation container.
[0213] The disclosure of Japanese Patent Application No.
2012-104816, filed on May 1, 2012, is incorporated herein by
reference in its entirety.
[0214] All the literature, patent applications, and technical
standards described herein are herein incorporated by reference to
the same extent as if each individual literature, patent
application, or technical standard was specifically and
individually indicated as being incorporated by reference.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 39 <210> SEQ ID NO 1 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Peptide
<400> SEQUENCE: 1 Cys Ser Tyr Tyr Gln Ser Cys 1 5 <210>
SEQ ID NO 2 <211> LENGTH: 32 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Heparin Binding Domain
<400> SEQUENCE: 2 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe
Arg His Arg Asn Arg 1 5 10 15 Lys Gly Tyr Arg Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn 20 25 30 <210> SEQ ID NO 3
<211> LENGTH: 459 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 Asp Gln Glu Ser Cys Lys Gly
Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys
Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr
Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe
Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr Asp Asp Gly Glu Glu 50 55
60 Lys Asn Asn Ala Thr Val His Glu Gln Val Gly Gly Pro Ser Leu Thr
65 70 75 80 Ser Asp Leu Gln Ala Gln Ser Lys Gly Asn Pro Glu Gln Thr
Pro Val 85 90 95 Leu Lys Pro Glu Glu Glu Ala Pro Ala Pro Glu Val
Gly Ala Ser Lys 100 105 110 Pro Glu Gly Ile Asp Ser Arg Pro Glu Thr
Leu His Pro Gly Arg Pro 115 120 125 Gln Pro Pro Ala Glu Glu Glu Leu
Cys Ser Gly Lys Pro Phe Asp Ala 130 135 140 Phe Thr Asp Leu Lys Asn
Gly Ser Leu Phe Ala Phe Arg Gly Gln Tyr 145 150 155 160 Cys Tyr Glu
Leu Asp Glu Lys Ala Val Arg Pro Gly Tyr Pro Lys Leu 165 170 175 Ile
Arg Asp Val Trp Gly Ile Glu Gly Pro Ile Asp Ala Ala Phe Thr 180 185
190 Arg Ile Asn Cys Gln Gly Lys Thr Tyr Leu Phe Lys Gly Ser Gln Tyr
195 200 205 Trp Arg Phe Glu Asp Gly Val Leu Asp Pro Asp Tyr Pro Arg
Asn Ile 210 215 220 Ser Asp Gly Phe Asp Gly Ile Pro Asp Asn Val Asp
Ala Ala Leu Ala 225 230 235 240 Leu Pro Ala His Ser Tyr Ser Gly Arg
Glu Arg Val Tyr Phe Phe Lys 245 250 255 Gly Lys Gln Tyr Trp Glu Tyr
Gln Phe Gln His Gln Pro Ser Gln Glu 260 265 270 Glu Cys Glu Gly Ser
Ser Leu Ser Ala Val Phe Glu His Phe Ala Met 275 280 285 Met Gln Arg
Asp Ser Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly 290 295 300 Arg
Thr Ser Ala Gly Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp 305 310
315 320 His Gly Val Pro Gly Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr 325 330 335 Ile Ser Gly Met Ala Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe 340 345 350 Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg 355 360 365 Gly Arg Asn Gln Asn Ser Arg Arg Pro
Ser Arg Ala Thr Trp Leu Ser 370 375 380 Leu Phe Ser Ser Glu Glu Ser
Asn Leu Gly Ala Asn Asn Tyr Asp Asp 385 390 395 400 Tyr Arg Met Asp
Trp Leu Val Pro Ala Thr Cys Glu Pro Ile Gln Ser 405 410 415 Val Phe
Phe Phe Ser Gly Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr 420 425 430
Arg Arg Val Asp Thr Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln 435
440 445 Tyr Trp Leu Gly Cys Pro Ala Pro Gly His Leu 450 455
<210> SEQ ID NO 4 <211> LENGTH: 246 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 4 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn
Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln
Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Pro
Ser Gln Glu Glu Cys Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val Phe
Glu His Phe Ala Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp Ile
Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr Arg
Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105 110
Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala 115
120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn
Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg
Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro Ser Arg Ala Thr Trp Leu
Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser Asn Leu Gly Ala Asn Asn
Tyr Asp Asp Tyr Arg Met Asp Trp 180 185 190 Leu Val Pro Ala Thr Cys
Glu Pro Ile Gln Ser Val Phe Phe Phe Ser 195 200 205 Gly Asp Lys Tyr
Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp Thr 210 215 220 Val Asp
Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly Cys 225 230 235
240 Pro Ala Pro Gly His Leu 245 <210> SEQ ID NO 5 <211>
LENGTH: 87 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide <400> SEQUENCE: 5 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Pro Arg Pro Ser Leu Ala Lys Lys Gln 50
55 60 Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln Arg Gly
His 65 70 75 80 Ser Arg Gly Arg Asn Gln Asn 85 <210> SEQ ID
NO 6 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide <400> SEQUENCE: 6 Asp
Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10
15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys
20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly
Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Gly Val Pro Gly Gln
Val Asp Ala Ala 50 55 60 Met Ala Gly Arg Ile Tyr Ile Ser Gly Met
Ala Pro Arg Pro Ser Leu 65 70 75 80 Ala Lys Lys Gln Arg Phe Arg His
Arg Asn Arg Lys Gly Tyr Arg Ser 85 90 95 Gln Arg Gly His Ser Arg
Gly Arg Asn Gln Asn 100 105 <210> SEQ ID NO 7 <211>
LENGTH: 117 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide <400> SEQUENCE: 7 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Gln Pro Gln Phe Ile Ser Arg Asp Trp 50
55 60 His Gly Val Pro Gly Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr 65 70 75 80 Ile Ser Gly Met Ala Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe 85 90 95 Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg 100 105 110 Gly Arg Asn Gln Asn 115 <210>
SEQ ID NO 8 <211> LENGTH: 127 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 8 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn
Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln
Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Phe
Trp Gly Arg Thr Ser Ala Gly Thr 50 55 60 Arg Gln Pro Gln Phe Ile
Ser Arg Asp Trp His Gly Val Pro Gly Gln 65 70 75 80 Val Asp Ala Ala
Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro 85 90 95 Arg Pro
Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys 100 105 110
Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 115 120
125 <210> SEQ ID NO 9 <211> LENGTH: 245 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Peptide
<400> SEQUENCE: 9 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu
Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys
Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys
Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu
Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60 Leu Ser Ala
Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65 70 75 80 Glu
Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr 85 90
95 Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly Gln
100 105 110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met
Ala Pro 115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His
Arg Asn Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg
Gly Arg Asn Gln Asn Ser 145 150 155 160 Arg Arg Pro Ser Arg Ala Thr
Trp Leu Ser Leu Phe Ser Ser Glu Glu 165 170 175 Ser Asn Leu Gly Ala
Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp Leu 180 185 190 Val Pro Ala
Thr Ser Glu Pro Ile Gln Ser Val Phe Phe Phe Ser Gly 195 200 205 Asp
Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp Thr Val 210 215
220 Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly Ser Pro
225 230 235 240 Ala Pro Gly His Leu 245 <210> SEQ ID NO 10
<211> LENGTH: 159 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 10 Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25
30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val
35 40 45 Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly
Ser Ser 50 55 60 Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln
Arg Asp Ser Trp 65 70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly
Arg Thr Ser Ala Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg
Asp Trp His Gly Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala
Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu
Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr
Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155
<210> SEQ ID NO 11 <211> LENGTH: 165 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 11 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe
Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr
Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro
Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu
Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60 Leu Ser Ala Val Phe
Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65 70 75 80 Glu Asp Ile
Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr 85 90 95 Arg
Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly Gln 100 105
110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro
115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn
Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg
Asn Gln Asn Ser 145 150 155 160 Arg Arg Pro Ser Arg 165 <210>
SEQ ID NO 12 <211> LENGTH: 175 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 12 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe
Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr
Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro
Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu
Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60 Leu Ser Ala Val Phe
Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65 70 75 80 Glu Asp Ile
Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr 85 90 95 Arg
Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly Gln 100 105
110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro
115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn
Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg
Asn Gln Asn Ser 145 150 155 160 Arg Arg Pro Ser Arg Ala Thr Trp Leu
Ser Leu Phe Ser Ser Glu 165 170 175 <210> SEQ ID NO 13
<211> LENGTH: 185 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 13 Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25
30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val
35 40 45 Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly
Ser Ser 50 55 60 Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln
Arg Asp Ser Trp 65 70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly
Arg Thr Ser Ala Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg
Asp Trp His Gly Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala
Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu
Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr
Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser 145 150 155
160 Arg Arg Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu Glu
165 170 175 Ser Asn Leu Gly Ala Asn Asn Tyr Asp 180 185 <210>
SEQ ID NO 14 <211> LENGTH: 247 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-1 <400>
SEQUENCE: 14 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp
Glu Pro Ser Gln Glu Glu Cys Glu Gly 50 55 60 Ser Ser Leu Ser Ala
Val Phe Glu His Phe Ala Met Met Gln Arg Asp 65 70 75 80 Ser Trp Glu
Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala 85 90 95 Gly
Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro 100 105
110 Gly Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met
115 120 125 Ala Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His
Arg Asn 130 135 140 Arg Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg
Gly Arg Asn Gln 145 150 155 160 Asn Ser Arg Arg Pro Ser Arg Ala Thr
Trp Leu Ser Leu Phe Ser Ser 165 170 175 Glu Glu Ser Asn Leu Gly Ala
Asn Asn Tyr Asp Asp Tyr Arg Met Asp 180 185 190 Trp Leu Val Pro Ala
Thr Cys Glu Pro Ile Gln Ser Val Phe Phe Phe 195 200 205 Ser Gly Asp
Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp 210 215 220 Thr
Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly 225 230
235 240 Cys Pro Ala Pro Gly His Leu 245 <210> SEQ ID NO 15
<211> LENGTH: 88 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-2 <400> SEQUENCE: 15 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Pro Arg Pro Ser
Leu Ala Lys Lys 50 55 60 Gln Arg Phe Arg His Arg Asn Arg Lys Gly
Tyr Arg Ser Gln Arg Gly 65 70 75 80 His Ser Arg Gly Arg Asn Gln Asn
85 <210> SEQ ID NO 16 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Peptide RCP-3
<400> SEQUENCE: 16 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys
Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu
Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala
Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met
Pro Glu Asp Glu Gly Val Pro Gly Gln Val Asp Ala 50 55 60 Ala Met
Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro Ser 65 70 75 80
Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg 85
90 95 Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 100 105
<210> SEQ ID NO 17 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-4 <400>
SEQUENCE: 17 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp
Glu Gln Pro Gln Phe Ile Ser Arg Asp 50 55 60 Trp His Gly Val Pro
Gly Gln Val Asp Ala Ala Met Ala Gly Arg Ile 65 70 75 80 Tyr Ile Ser
Gly Met Ala Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg 85 90 95 Phe
Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln Arg Gly His Ser 100 105
110 Arg Gly Arg Asn Gln Asn 115 <210> SEQ ID NO 18
<211> LENGTH: 128 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-5 <400> SEQUENCE: 18 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Phe Trp Gly Arg
Thr Ser Ala Gly 50 55 60 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp
Trp His Gly Val Pro Gly 65 70 75 80 Gln Val Asp Ala Ala Met Ala Gly
Arg Ile Tyr Ile Ser Gly Met Ala 85 90 95 Pro Arg Pro Ser Leu Ala
Lys Lys Gln Arg Phe Arg His Arg Asn Arg 100 105 110 Lys Gly Tyr Arg
Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 115 120 125
<210> SEQ ID NO 19 <211> LENGTH: 246 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-6 <400>
SEQUENCE: 19 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp
Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val
Phe Glu His Phe Ala Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp
Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr
Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105
110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala
115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg
Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly
Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro Ser Arg Ala Thr Trp
Leu Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser Asn Leu Gly Ala Asn
Asn Tyr Asp Asp Tyr Arg Met Asp Trp 180 185 190 Leu Val Pro Ala Thr
Ser Glu Pro Ile Gln Ser Val Phe Phe Phe Ser 195 200 205 Gly Asp Lys
Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp Thr 210 215 220 Val
Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly Ser 225 230
235 240 Pro Ala Pro Gly His Leu 245 <210> SEQ ID NO 20
<211> LENGTH: 160 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-7 <400> SEQUENCE: 20 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu
Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val Phe Glu His Phe Ala
Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp Ile Phe Glu Leu Leu
Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr Arg Gln Pro Gln Phe
Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105 110 Gln Val Asp Ala
Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala 115 120 125 Pro Arg
Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg 130 135 140
Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145
150 155 160 <210> SEQ ID NO 21 <211> LENGTH: 166
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-8 <400> SEQUENCE: 21 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55
60 Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
65 70 75 80 Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly 85 90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg
Pro Ser Arg 165 <210> SEQ ID NO 22 <211> LENGTH: 176
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-9 <400> SEQUENCE: 22 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55
60 Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
65 70 75 80 Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly 85 90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg
Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu 165 170 175
<210> SEQ ID NO 23 <211> LENGTH: 186 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-10 <400>
SEQUENCE: 23 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp
Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val
Phe Glu His Phe Ala Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp
Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr
Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105
110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala
115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg
Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly
Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro Ser Arg Ala Thr Trp
Leu Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser Asn Leu Gly Ala Asn
Asn Tyr Asp 180 185 <210> SEQ ID NO 24 <211> LENGTH:
463 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide CRP-11 <400> SEQUENCE: 24 Gly Pro Leu Gly Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn Val Asp Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30 Gln Ser Cys
Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35 40 45 Arg
Gly Asp Val Phe Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr Asp 50 55
60 Asp Gly Glu Glu Lys Asn Asn Ala Thr Val His Glu Gln Val Gly Gly
65 70 75 80 Pro Ser Leu Thr Ser Asp Leu Gln Ala Gln Ser Lys Gly Asn
Pro Glu 85 90 95 Gln Thr Pro Val Leu Lys Pro Glu Glu Glu Ala Pro
Ala Pro Glu Val 100 105 110 Gly Ala Ser Lys Pro Glu Gly Ile Asp Ser
Arg Pro Glu Thr Leu His 115 120 125 Pro Gly Arg Pro Gln Pro Pro Ala
Glu Glu Glu Leu Cys Ser Gly Lys 130 135 140 Pro Phe Asp Ala Phe Thr
Asp Leu Lys Asn Gly Ser Leu Phe Ala Phe 145 150 155 160 Arg Gly Gln
Tyr Cys Tyr Glu Leu Asp Glu Lys Ala Val Arg Pro Gly 165 170 175 Tyr
Pro Lys Leu Ile Arg Asp Val Trp Gly Ile Glu Gly Pro Ile Asp 180 185
190 Ala Ala Phe Thr Arg Ile Asn Cys Gln Gly Lys Thr Tyr Leu Phe Lys
195 200 205 Gly Ser Gln Tyr Trp Arg Phe Glu Asp Gly Val Leu Asp Pro
Asp Tyr 210 215 220 Pro Arg Asn Ile Ser Asp Gly Phe Asp Gly Ile Pro
Asp Asn Val Asp 225 230 235 240 Ala Ala Leu Ala Leu Pro Ala His Ser
Tyr Ser Gly Arg Glu Arg Val 245 250 255 Tyr Phe Phe Lys Gly Lys Gln
Tyr Trp Glu Tyr Gln Phe Gln His Gln 260 265 270 Pro Ser Gln Glu Glu
Cys Glu Gly Ser Ser Leu Ser Ala Val Phe Glu 275 280 285 His Phe Ala
Met Met Gln Arg Asp Ser Trp Glu Asp Ile Phe Glu Leu 290 295 300 Leu
Phe Trp Gly Arg Thr Ser Ala Gly Thr Arg Gln Pro Gln Phe Ile 305 310
315 320 Ser Arg Asp Trp His Gly Val Pro Gly Gln Val Asp Ala Ala Met
Ala 325 330 335 Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro Ser
Leu Ala Lys 340 345 350 Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly
Tyr Arg Ser Gln Arg 355 360 365 Gly His Ser Arg Gly Arg Asn Gln Asn
Ser Arg Arg Pro Ser Arg Ala 370 375 380 Thr Trp Leu Ser Leu Phe Ser
Ser Glu Glu Ser Asn Leu Gly Ala Asn 385 390 395 400 Asn Tyr Asp Asp
Tyr Arg Met Asp Trp Leu Val Pro Ala Thr Cys Glu 405 410 415 Pro Ile
Gln Ser Val Phe Phe Phe Ser Gly Asp Lys Tyr Tyr Arg Val 420 425 430
Asn Leu Arg Thr Arg Arg Val Asp Thr Val Asp Pro Pro Tyr Pro Arg 435
440 445 Ser Ile Ala Gln Tyr Trp Leu Gly Cys Pro Ala Pro Gly His Leu
450 455 460 <210> SEQ ID NO 25 <211> LENGTH: 272
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-12 <400> SEQUENCE: 25 Gly Pro Leu Gly Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn Val Asp Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30 Gln Ser Cys
Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35 40 45 Arg
Gly Asp Val Phe Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr Asp 50 55
60 Asp Gly Glu Glu Lys Asn Asn Ala Thr Val His Glu Gln Val Gly Gly
65 70 75 80 Pro Ser Leu Thr Ser Asp Leu Gln Ala Gln Ser Lys Gly Asn
Pro Glu 85 90 95 Gln Thr Pro Val Leu Lys Pro Glu Glu Glu Ala Pro
Ala Pro Glu Val 100 105 110 Gly Ala Ser Lys Pro Glu Gly Ile Asp Ser
Arg Pro Glu Thr Leu His 115 120 125 Pro Gly Arg Pro Gln Pro Pro Ala
Glu Glu Glu Leu Cys Ser Gly Lys 130 135 140 Pro Phe Asp Ala Phe Thr
Asp Leu Lys Asn Gly Ser Leu Phe Ala Phe 145 150 155 160 Arg Gly Gln
Tyr Cys Tyr Glu Leu Asp Glu Lys Ala Val Arg Pro Gly 165 170 175 Tyr
Pro Lys Leu Ile Arg Asp Val Trp Gly Ile Glu Gly Pro Ile Asp 180 185
190 Ala Ala Phe Thr Arg Ile Asn Cys Gln Gly Lys Thr Tyr Leu Phe Lys
195 200 205 Gly Ser Gln Tyr Trp Arg Phe Glu Asp Gly Val Leu Asp Pro
Asp Tyr 210 215 220 Pro Arg Asn Ile Ser Asp Gly Phe Asp Gly Ile Pro
Asp Asn Val Asp 225 230 235 240 Ala Ala Leu Ala Leu Pro Ala His Ser
Tyr Ser Gly Arg Glu Arg Val 245 250 255 Tyr Phe Phe Lys Gly Lys Gln
Tyr Trp Glu Tyr Gln Phe Gln His Gln 260 265 270 <210> SEQ ID
NO 26 <211> LENGTH: 134 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide RCP-13 <400> SEQUENCE:
26 Gly Pro Leu Gly Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
1 5 10 15 Phe Asn Val Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr 20 25 30 Gln Ser Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr 35 40 45 Arg Gly Asp Val Phe Thr Met Pro Glu Asp
Glu Tyr Thr Val Tyr Asp 50 55 60 Asp Gly Glu Glu Lys Asn Asn Ala
Thr Val His Glu Gln Val Gly Gly 65 70 75 80 Pro Ser Leu Thr Ser Asp
Leu Gln Ala Gln Ser Lys Gly Asn Pro Glu 85 90 95 Gln Thr Pro Val
Leu Lys Pro Glu Glu Glu Ala Pro Ala Pro Glu Val 100 105 110 Gly Ala
Ser Lys Pro Glu Gly Ile Asp Ser Arg Pro Glu Thr Leu His 115 120 125
Pro Gly Arg Pro Gln Pro 130 <210> SEQ ID NO 27 <211>
LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide RCP-14 <400> SEQUENCE: 27 Gly Pro Leu Gly
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn
Val Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30
Gln Ser Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35
40 45 Arg Gly Asp Val Phe Thr Met Pro Glu Asp Glu 50 55 <210>
SEQ ID NO 28 <211> LENGTH: 408 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-15 <400>
SEQUENCE: 28 Gly Pro Leu Gly Tyr Thr Val Tyr Asp Asp Gly Glu Glu
Lys Asn Asn 1 5 10 15 Ala Thr Val His Glu Gln Val Gly Gly Pro Ser
Leu Thr Ser Asp Leu 20 25 30 Gln Ala Gln Ser Lys Gly Asn Pro Glu
Gln Thr Pro Val Leu Lys Pro 35 40 45 Glu Glu Glu Ala Pro Ala Pro
Glu Val Gly Ala Ser Lys Pro Glu Gly 50 55 60 Ile Asp Ser Arg Pro
Glu Thr Leu His Pro Gly Arg Pro Gln Pro Pro 65 70 75 80 Ala Glu Glu
Glu Leu Cys Ser Gly Lys Pro Phe Asp Ala Phe Thr Asp 85 90 95 Leu
Lys Asn Gly Ser Leu Phe Ala Phe Arg Gly Gln Tyr Cys Tyr Glu 100 105
110 Leu Asp Glu Lys Ala Val Arg Pro Gly Tyr Pro Lys Leu Ile Arg Asp
115 120 125 Val Trp Gly Ile Glu Gly Pro Ile Asp Ala Ala Phe Thr Arg
Ile Asn 130 135 140 Cys Gln Gly Lys Thr Tyr Leu Phe Lys Gly Ser Gln
Tyr Trp Arg Phe 145 150 155 160 Glu Asp Gly Val Leu Asp Pro Asp Tyr
Pro Arg Asn Ile Ser Asp Gly 165 170 175 Phe Asp Gly Ile Pro Asp Asn
Val Asp Ala Ala Leu Ala Leu Pro Ala 180 185 190 His Ser Tyr Ser Gly
Arg Glu Arg Val Tyr Phe Phe Lys Gly Lys Gln 195 200 205 Tyr Trp Glu
Tyr Gln Phe Gln His Gln Pro Ser Gln Glu Glu Cys Glu 210 215 220 Gly
Ser Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg 225 230
235 240 Asp Ser Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr
Ser 245 250 255 Ala Gly Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp
His Gly Val 260 265 270 Pro Gly Gln Val Asp Ala Ala Met Ala Gly Arg
Ile Tyr Ile Ser Gly 275 280 285 Met Ala Pro Arg Pro Ser Leu Ala Lys
Lys Gln Arg Phe Arg His Arg 290 295 300 Asn Arg Lys Gly Tyr Arg Ser
Gln Arg Gly His Ser Arg Gly Arg Asn 305 310 315 320 Gln Asn Ser Arg
Arg Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser 325 330 335 Ser Glu
Glu Ser Asn Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met 340 345 350
Asp Trp Leu Val Pro Ala Thr Cys Glu Pro Ile Gln Ser Val Phe Phe 355
360 365 Phe Ser Gly Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg
Val 370 375 380 Asp Thr Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln
Tyr Trp Leu 385 390 395 400 Gly Cys Pro Ala Pro Gly His Leu 405
<210> SEQ ID NO 29 <211> LENGTH: 195 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-16 <400>
SEQUENCE: 29 Gly Pro Leu Gly Pro Ser Gln Glu Glu Cys Glu Gly Ser
Ser Leu Ser 1 5 10 15 Ala Val Phe Glu His Phe Ala Met Met Gln Arg
Asp Ser Trp Glu Asp 20 25 30 Ile Phe Glu Leu Leu Phe Trp Gly Arg
Thr Ser Ala Gly Thr Arg Gln 35 40 45 Pro Gln Phe Ile Ser Arg Asp
Trp His Gly Val Pro Gly Gln Val Asp 50 55 60 Ala Ala Met Ala Gly
Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro 65 70 75 80 Ser Leu Ala
Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr 85 90 95 Arg
Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser Arg Arg 100 105
110 Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu Glu Ser Asn
115 120 125 Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp Leu
Val Pro 130 135 140 Ala Thr Cys Glu Pro Ile Gln Ser Val Phe Phe Phe
Ser Gly Asp Lys 145 150 155 160 Tyr Tyr Arg Val Asn Leu Arg Thr Arg
Arg Val Asp Thr Val Asp Pro 165 170 175 Pro Tyr Pro Arg Ser Ile Ala
Gln Tyr Trp Leu Gly Cys Pro Ala Pro 180 185 190 Gly His Leu 195
<210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 30 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe
Asn Val Asp 1 5 10 15 Lys Lys Cys Gln 20 <210> SEQ ID NO 31
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 31 Lys Gly Arg
Cys Thr Glu Gly Phe Asn Val Asp Lys Lys Cys Gln Cys 1 5 10 15 Asp
Glu Leu Cys 20 <210> SEQ ID NO 32 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 32 Glu Gly Phe Asn Val Asp Lys Lys
Cys Gln Cys Asp Glu Leu Cys Ser 1 5 10 15 Tyr Tyr Gln Ser 20
<210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 33 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys 1 5 10 15 Cys Thr Asp Tyr 20 <210> SEQ ID NO 34
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 34 Cys Cys Thr
Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly 1 5 10 15 Asp
Val Phe Thr 20 <210> SEQ ID NO 35 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 35 Thr Ala Glu Cys Lys Pro Gln Val
Thr Arg Gly Asp Val Phe Thr Met 1 5 10 15 Pro Glu Asp Glu 20
<210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 36 Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val
Thr Arg Gly 1 5 10 15 Glu Val Phe Thr 20 <210> SEQ ID NO 37
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 37 Thr Ala Glu
Cys Lys Pro Gln Val Thr Arg Gly Glu Val Phe Thr Met 1 5 10 15 Pro
Glu Asp Glu 20 <210> SEQ ID NO 38 <211> LENGTH: 142
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-17aa <400> SEQUENCE: 38 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Glu 50
55 60 Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr
Arg 65 70 75 80 Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro
Gly Gln Val 85 90 95 Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser
Gly Met Ala Pro Arg 100 105 110 Pro Ser Leu Ala Lys Lys Gln Arg Phe
Arg His Arg Asn Arg Lys Gly 115 120 125 Tyr Arg Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn 130 135 140 <210> SEQ ID NO 39
<211> LENGTH: 143 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-17 <400> SEQUENCE: 39 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu
Ser Glu Gly Ser 50 55 60 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly
Arg Thr Ser Ala Gly Thr 65 70 75 80 Arg Gln Pro Gln Phe Ile Ser Arg
Asp Trp His Gly Val Pro Gly Gln 85 90 95 Val Asp Ala Ala Met Ala
Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro 100 105 110 Arg Pro Ser Leu
Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys 115 120 125 Gly Tyr
Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 130 135 140
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210>
SEQ ID NO 1 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide <400> SEQUENCE: 1 Cys
Ser Tyr Tyr Gln Ser Cys 1 5 <210> SEQ ID NO 2 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Heparin Binding Domain <400> SEQUENCE: 2 Pro Arg
Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg 1 5 10 15
Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 20
25 30 <210> SEQ ID NO 3 <211> LENGTH: 459 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
3 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1
5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr
Asp Asp Gly Glu Glu 50 55 60 Lys Asn Asn Ala Thr Val His Glu Gln
Val Gly Gly Pro Ser Leu Thr 65 70 75 80 Ser Asp Leu Gln Ala Gln Ser
Lys Gly Asn Pro Glu Gln Thr Pro Val 85 90 95 Leu Lys Pro Glu Glu
Glu Ala Pro Ala Pro Glu Val Gly Ala Ser Lys 100 105 110 Pro Glu Gly
Ile Asp Ser Arg Pro Glu Thr Leu His Pro Gly Arg Pro 115 120 125 Gln
Pro Pro Ala Glu Glu Glu Leu Cys Ser Gly Lys Pro Phe Asp Ala 130 135
140 Phe Thr Asp Leu Lys Asn Gly Ser Leu Phe Ala Phe Arg Gly Gln Tyr
145 150 155 160 Cys Tyr Glu Leu Asp Glu Lys Ala Val Arg Pro Gly Tyr
Pro Lys Leu 165 170 175 Ile Arg Asp Val Trp Gly Ile Glu Gly Pro Ile
Asp Ala Ala Phe Thr 180 185 190 Arg Ile Asn Cys Gln Gly Lys Thr Tyr
Leu Phe Lys Gly Ser Gln Tyr 195 200 205 Trp Arg Phe Glu Asp Gly Val
Leu Asp Pro Asp Tyr Pro Arg Asn Ile 210 215 220 Ser Asp Gly Phe Asp
Gly Ile Pro Asp Asn Val Asp Ala Ala Leu Ala 225 230 235 240 Leu Pro
Ala His Ser Tyr Ser Gly Arg Glu Arg Val Tyr Phe Phe Lys 245 250 255
Gly Lys Gln Tyr Trp Glu Tyr Gln Phe Gln His Gln Pro Ser Gln Glu 260
265 270 Glu Cys Glu Gly Ser Ser Leu Ser Ala Val Phe Glu His Phe Ala
Met 275 280 285 Met Gln Arg Asp Ser Trp Glu Asp Ile Phe Glu Leu Leu
Phe Trp Gly 290 295 300 Arg Thr Ser Ala Gly Thr Arg Gln Pro Gln Phe
Ile Ser Arg Asp Trp 305 310 315 320 His Gly Val Pro Gly Gln Val Asp
Ala Ala Met Ala Gly Arg Ile Tyr 325 330 335 Ile Ser Gly Met Ala Pro
Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe 340 345 350 Arg His Arg Asn
Arg Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg 355 360 365 Gly Arg
Asn Gln Asn Ser Arg Arg Pro Ser Arg Ala Thr Trp Leu Ser 370 375 380
Leu Phe Ser Ser Glu Glu Ser Asn Leu Gly Ala Asn Asn Tyr Asp Asp 385
390 395 400 Tyr Arg Met Asp Trp Leu Val Pro Ala Thr Cys Glu Pro Ile
Gln Ser 405 410 415 Val Phe Phe Phe Ser Gly Asp Lys Tyr Tyr Arg Val
Asn Leu Arg Thr 420 425 430 Arg Arg Val Asp Thr Val Asp Pro Pro Tyr
Pro Arg Ser Ile Ala Gln 435 440 445 Tyr Trp Leu Gly Cys Pro Ala Pro
Gly His Leu 450 455 <210> SEQ ID NO 4 <211> LENGTH: 246
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 4 Asp Gln Glu Ser Cys Lys Gly Arg Cys
Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu
Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala
Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met
Pro Glu Asp Glu Pro Ser Gln Glu Glu Cys Glu Gly Ser 50 55 60 Ser
Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser 65 70
75 80 Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala
Gly 85 90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly
Val Pro Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr
Ile Ser Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln
Arg Phe Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg
Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro
Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser
Asn Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp 180 185 190
Leu Val Pro Ala Thr Cys Glu Pro Ile Gln Ser Val Phe Phe Phe Ser 195
200 205 Gly Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp
Thr 210 215 220 Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp
Leu Gly Cys 225 230 235 240 Pro Ala Pro Gly His Leu 245 <210>
SEQ ID NO 5 <211> LENGTH: 87 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 5 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn
Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln
Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Pro
Arg Pro Ser Leu Ala Lys Lys Gln 50 55 60 Arg Phe Arg His Arg Asn
Arg Lys Gly Tyr Arg Ser Gln Arg Gly His 65 70 75 80 Ser Arg Gly Arg
Asn Gln Asn 85 <210> SEQ ID NO 6 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 6 Asp Gln Glu Ser Cys Lys Gly Arg Cys
Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu
Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala
Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met
Pro Glu Asp Glu Gly Val Pro Gly Gln Val Asp Ala Ala 50 55 60 Met
Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro Ser Leu 65 70
75 80 Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg
Ser 85 90 95 Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn 100 105
<210> SEQ ID NO 7 <211> LENGTH: 117 <212> TYPE:
PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 7 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn
Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln
Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu Gln
Pro Gln Phe Ile Ser Arg Asp Trp 50 55 60 His Gly Val Pro Gly Gln
Val Asp Ala Ala Met Ala Gly Arg Ile Tyr 65 70 75 80 Ile Ser Gly Met
Ala Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe 85 90 95 Arg His
Arg Asn Arg Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg 100 105 110
Gly Arg Asn Gln Asn 115 <210> SEQ ID NO 8 <211> LENGTH:
127 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 8 Asp Gln Glu Ser Cys Lys Gly Arg Cys
Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu
Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala
Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met
Pro Glu Asp Glu Phe Trp Gly Arg Thr Ser Ala Gly Thr 50 55 60 Arg
Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly Gln 65 70
75 80 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala
Pro 85 90 95 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg
Asn Arg Lys 100 105 110 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly
Arg Asn Gln Asn 115 120 125 <210> SEQ ID NO 9 <211>
LENGTH: 245 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide <400> SEQUENCE: 9 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Ser 50
55 60 Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
Trp 65 70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser 145 150 155 160 Arg Arg
Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu Glu 165 170 175
Ser Asn Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp Leu 180
185 190 Val Pro Ala Thr Ser Glu Pro Ile Gln Ser Val Phe Phe Phe Ser
Gly 195 200 205 Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val
Asp Thr Val 210 215 220 Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr
Trp Leu Gly Ser Pro 225 230 235 240 Ala Pro Gly His Leu 245
<210> SEQ ID NO 10 <211> LENGTH: 159 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 10 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe
Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr
Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys Lys Pro
Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr Met Pro Glu Asp Glu
Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60 Leu Ser Ala Val Phe
Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65 70 75 80 Glu Asp Ile
Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr 85 90 95 Arg
Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly Gln 100 105
110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro
115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn
Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg
Asn Gln Asn 145 150 155 <210> SEQ ID NO 11 <211>
LENGTH: 165 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide <400> SEQUENCE: 11 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Ser 50
55 60 Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
Trp 65 70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser 145 150 155 160 Arg Arg
Pro Ser Arg 165 <210> SEQ ID NO 12 <211> LENGTH: 175
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 12 Asp Gln Glu Ser Cys Lys Gly Arg
Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp
Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr
Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr
Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60
Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65
70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala
Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly
Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr
Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln
Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg
Gly His Ser Arg Gly Arg Asn Gln Asn Ser 145 150 155 160 Arg Arg Pro
Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu 165 170 175
<210> SEQ ID NO 13 <211> LENGTH: 185
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 13 Asp Gln Glu Ser Cys Lys Gly Arg
Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp
Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr
Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45 Phe Thr
Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Ser 50 55 60
Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser Trp 65
70 75 80 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala
Gly Thr 85 90 95 Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly
Val Pro Gly Gln 100 105 110 Val Asp Ala Ala Met Ala Gly Arg Ile Tyr
Ile Ser Gly Met Ala Pro 115 120 125 Arg Pro Ser Leu Ala Lys Lys Gln
Arg Phe Arg His Arg Asn Arg Lys 130 135 140 Gly Tyr Arg Ser Gln Arg
Gly His Ser Arg Gly Arg Asn Gln Asn Ser 145 150 155 160 Arg Arg Pro
Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu Glu 165 170 175 Ser
Asn Leu Gly Ala Asn Asn Tyr Asp 180 185 <210> SEQ ID NO 14
<211> LENGTH: 247 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-1 <400> SEQUENCE: 14 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Pro Ser Gln Glu
Glu Cys Glu Gly 50 55 60 Ser Ser Leu Ser Ala Val Phe Glu His Phe
Ala Met Met Gln Arg Asp 65 70 75 80 Ser Trp Glu Asp Ile Phe Glu Leu
Leu Phe Trp Gly Arg Thr Ser Ala 85 90 95 Gly Thr Arg Gln Pro Gln
Phe Ile Ser Arg Asp Trp His Gly Val Pro 100 105 110 Gly Gln Val Asp
Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met 115 120 125 Ala Pro
Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn 130 135 140
Arg Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln 145
150 155 160 Asn Ser Arg Arg Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe
Ser Ser 165 170 175 Glu Glu Ser Asn Leu Gly Ala Asn Asn Tyr Asp Asp
Tyr Arg Met Asp 180 185 190 Trp Leu Val Pro Ala Thr Cys Glu Pro Ile
Gln Ser Val Phe Phe Phe 195 200 205 Ser Gly Asp Lys Tyr Tyr Arg Val
Asn Leu Arg Thr Arg Arg Val Asp 210 215 220 Thr Val Asp Pro Pro Tyr
Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly 225 230 235 240 Cys Pro Ala
Pro Gly His Leu 245 <210> SEQ ID NO 15 <211> LENGTH: 88
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-2 <400> SEQUENCE: 15 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Pro Arg Pro Ser Leu Ala Lys Lys 50 55
60 Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln Arg Gly
65 70 75 80 His Ser Arg Gly Arg Asn Gln Asn 85 <210> SEQ ID
NO 16 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide RCP-3 <400> SEQUENCE: 16
Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5
10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser
Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr
Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Gly Val Pro
Gly Gln Val Asp Ala 50 55 60 Ala Met Ala Gly Arg Ile Tyr Ile Ser
Gly Met Ala Pro Arg Pro Ser 65 70 75 80 Leu Ala Lys Lys Gln Arg Phe
Arg His Arg Asn Arg Lys Gly Tyr Arg 85 90 95 Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn 100 105 <210> SEQ ID NO 17
<211> LENGTH: 118 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-4 <400> SEQUENCE: 17 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Gln Pro Gln Phe
Ile Ser Arg Asp 50 55 60 Trp His Gly Val Pro Gly Gln Val Asp Ala
Ala Met Ala Gly Arg Ile 65 70 75 80 Tyr Ile Ser Gly Met Ala Pro Arg
Pro Ser Leu Ala Lys Lys Gln Arg 85 90 95 Phe Arg His Arg Asn Arg
Lys Gly Tyr Arg Ser Gln Arg Gly His Ser 100 105 110 Arg Gly Arg Asn
Gln Asn 115 <210> SEQ ID NO 18 <211> LENGTH: 128
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-5 <400> SEQUENCE: 18 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Phe Trp Gly Arg Thr Ser Ala Gly 50 55
60 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly
65 70 75 80 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly
Met Ala 85 90 95 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg
His Arg Asn Arg 100 105 110 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser
Arg Gly Arg Asn Gln Asn 115 120 125 <210> SEQ ID NO 19
<211> LENGTH: 246 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-6 <400> SEQUENCE: 19 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu
Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val Phe Glu His Phe Ala
Met Met Gln Arg Asp Ser 65 70 75 80
Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly 85
90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro
Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser
Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe
Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro Ser Arg
Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser Asn Leu
Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp 180 185 190 Leu Val
Pro Ala Thr Ser Glu Pro Ile Gln Ser Val Phe Phe Phe Ser 195 200 205
Gly Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg Val Asp Thr 210
215 220 Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr Trp Leu Gly
Ser 225 230 235 240 Pro Ala Pro Gly His Leu 245 <210> SEQ ID
NO 20 <211> LENGTH: 160 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide RCP-7 <400> SEQUENCE: 20
Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5
10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser
Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr
Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Ser Gln Glu
Glu Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val Phe Glu His Phe
Ala Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp Ile Phe Glu Leu
Leu Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr Arg Gln Pro Gln
Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105 110 Gln Val Asp
Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala 115 120 125 Pro
Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn Arg 130 135
140 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn
145 150 155 160 <210> SEQ ID NO 21 <211> LENGTH: 166
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-8 <400> SEQUENCE: 21 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55
60 Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
65 70 75 80 Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly 85 90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg
Pro Ser Arg 165 <210> SEQ ID NO 22 <211> LENGTH: 176
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-9 <400> SEQUENCE: 22 Met Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp 35 40 45 Val
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55
60 Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg Asp Ser
65 70 75 80 Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser
Ala Gly 85 90 95 Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His
Gly Val Pro Gly 100 105 110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile
Tyr Ile Ser Gly Met Ala 115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys
Gln Arg Phe Arg His Arg Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln
Arg Gly His Ser Arg Gly Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg
Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu 165 170 175
<210> SEQ ID NO 23 <211> LENGTH: 186 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-10 <400>
SEQUENCE: 23 Met Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
Phe Asn Val 1 5 10 15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr Gln Ser Cys 20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr Arg Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp
Glu Ser Gln Glu Glu Ser Glu Gly Ser 50 55 60 Ser Leu Ser Ala Val
Phe Glu His Phe Ala Met Met Gln Arg Asp Ser 65 70 75 80 Trp Glu Asp
Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly 85 90 95 Thr
Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro Gly 100 105
110 Gln Val Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser Gly Met Ala
115 120 125 Pro Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg
Asn Arg 130 135 140 Lys Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly
Arg Asn Gln Asn 145 150 155 160 Ser Arg Arg Pro Ser Arg Ala Thr Trp
Leu Ser Leu Phe Ser Ser Glu 165 170 175 Glu Ser Asn Leu Gly Ala Asn
Asn Tyr Asp 180 185 <210> SEQ ID NO 24 <211> LENGTH:
463 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide CRP-11 <400> SEQUENCE: 24 Gly Pro Leu Gly Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn Val Asp Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30 Gln Ser Cys
Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35 40 45 Arg
Gly Asp Val Phe Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr Asp 50 55
60 Asp Gly Glu Glu Lys Asn Asn Ala Thr Val His Glu Gln Val Gly Gly
65 70 75 80 Pro Ser Leu Thr Ser Asp Leu Gln Ala Gln Ser Lys Gly Asn
Pro Glu 85 90 95 Gln Thr Pro Val Leu Lys Pro Glu Glu Glu Ala Pro
Ala Pro Glu Val 100 105 110 Gly Ala Ser Lys Pro Glu Gly Ile Asp Ser
Arg Pro Glu Thr Leu His 115 120 125 Pro Gly Arg Pro Gln Pro Pro Ala
Glu Glu Glu Leu Cys Ser Gly Lys 130 135 140 Pro Phe Asp Ala Phe Thr
Asp Leu Lys Asn Gly Ser Leu Phe Ala Phe 145 150 155 160 Arg Gly Gln
Tyr Cys Tyr Glu Leu Asp Glu Lys Ala Val Arg Pro Gly 165 170 175 Tyr
Pro Lys Leu Ile Arg Asp Val Trp Gly Ile Glu Gly Pro Ile Asp 180 185
190
Ala Ala Phe Thr Arg Ile Asn Cys Gln Gly Lys Thr Tyr Leu Phe Lys 195
200 205 Gly Ser Gln Tyr Trp Arg Phe Glu Asp Gly Val Leu Asp Pro Asp
Tyr 210 215 220 Pro Arg Asn Ile Ser Asp Gly Phe Asp Gly Ile Pro Asp
Asn Val Asp 225 230 235 240 Ala Ala Leu Ala Leu Pro Ala His Ser Tyr
Ser Gly Arg Glu Arg Val 245 250 255 Tyr Phe Phe Lys Gly Lys Gln Tyr
Trp Glu Tyr Gln Phe Gln His Gln 260 265 270 Pro Ser Gln Glu Glu Cys
Glu Gly Ser Ser Leu Ser Ala Val Phe Glu 275 280 285 His Phe Ala Met
Met Gln Arg Asp Ser Trp Glu Asp Ile Phe Glu Leu 290 295 300 Leu Phe
Trp Gly Arg Thr Ser Ala Gly Thr Arg Gln Pro Gln Phe Ile 305 310 315
320 Ser Arg Asp Trp His Gly Val Pro Gly Gln Val Asp Ala Ala Met Ala
325 330 335 Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro Ser Leu
Ala Lys 340 345 350 Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr
Arg Ser Gln Arg 355 360 365 Gly His Ser Arg Gly Arg Asn Gln Asn Ser
Arg Arg Pro Ser Arg Ala 370 375 380 Thr Trp Leu Ser Leu Phe Ser Ser
Glu Glu Ser Asn Leu Gly Ala Asn 385 390 395 400 Asn Tyr Asp Asp Tyr
Arg Met Asp Trp Leu Val Pro Ala Thr Cys Glu 405 410 415 Pro Ile Gln
Ser Val Phe Phe Phe Ser Gly Asp Lys Tyr Tyr Arg Val 420 425 430 Asn
Leu Arg Thr Arg Arg Val Asp Thr Val Asp Pro Pro Tyr Pro Arg 435 440
445 Ser Ile Ala Gln Tyr Trp Leu Gly Cys Pro Ala Pro Gly His Leu 450
455 460 <210> SEQ ID NO 25 <211> LENGTH: 272
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-12 <400> SEQUENCE: 25 Gly Pro Leu Gly Asp Gln Glu
Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn Val Asp Lys
Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30 Gln Ser Cys
Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35 40 45 Arg
Gly Asp Val Phe Thr Met Pro Glu Asp Glu Tyr Thr Val Tyr Asp 50 55
60 Asp Gly Glu Glu Lys Asn Asn Ala Thr Val His Glu Gln Val Gly Gly
65 70 75 80 Pro Ser Leu Thr Ser Asp Leu Gln Ala Gln Ser Lys Gly Asn
Pro Glu 85 90 95 Gln Thr Pro Val Leu Lys Pro Glu Glu Glu Ala Pro
Ala Pro Glu Val 100 105 110 Gly Ala Ser Lys Pro Glu Gly Ile Asp Ser
Arg Pro Glu Thr Leu His 115 120 125 Pro Gly Arg Pro Gln Pro Pro Ala
Glu Glu Glu Leu Cys Ser Gly Lys 130 135 140 Pro Phe Asp Ala Phe Thr
Asp Leu Lys Asn Gly Ser Leu Phe Ala Phe 145 150 155 160 Arg Gly Gln
Tyr Cys Tyr Glu Leu Asp Glu Lys Ala Val Arg Pro Gly 165 170 175 Tyr
Pro Lys Leu Ile Arg Asp Val Trp Gly Ile Glu Gly Pro Ile Asp 180 185
190 Ala Ala Phe Thr Arg Ile Asn Cys Gln Gly Lys Thr Tyr Leu Phe Lys
195 200 205 Gly Ser Gln Tyr Trp Arg Phe Glu Asp Gly Val Leu Asp Pro
Asp Tyr 210 215 220 Pro Arg Asn Ile Ser Asp Gly Phe Asp Gly Ile Pro
Asp Asn Val Asp 225 230 235 240 Ala Ala Leu Ala Leu Pro Ala His Ser
Tyr Ser Gly Arg Glu Arg Val 245 250 255 Tyr Phe Phe Lys Gly Lys Gln
Tyr Trp Glu Tyr Gln Phe Gln His Gln 260 265 270 <210> SEQ ID
NO 26 <211> LENGTH: 134 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Peptide RCP-13 <400> SEQUENCE:
26 Gly Pro Leu Gly Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly
1 5 10 15 Phe Asn Val Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser
Tyr Tyr 20 25 30 Gln Ser Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys
Pro Gln Val Thr 35 40 45 Arg Gly Asp Val Phe Thr Met Pro Glu Asp
Glu Tyr Thr Val Tyr Asp 50 55 60 Asp Gly Glu Glu Lys Asn Asn Ala
Thr Val His Glu Gln Val Gly Gly 65 70 75 80 Pro Ser Leu Thr Ser Asp
Leu Gln Ala Gln Ser Lys Gly Asn Pro Glu 85 90 95 Gln Thr Pro Val
Leu Lys Pro Glu Glu Glu Ala Pro Ala Pro Glu Val 100 105 110 Gly Ala
Ser Lys Pro Glu Gly Ile Asp Ser Arg Pro Glu Thr Leu His 115 120 125
Pro Gly Arg Pro Gln Pro 130 <210> SEQ ID NO 27 <211>
LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Peptide RCP-14 <400> SEQUENCE: 27 Gly Pro Leu Gly
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly 1 5 10 15 Phe Asn
Val Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr 20 25 30
Gln Ser Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr 35
40 45 Arg Gly Asp Val Phe Thr Met Pro Glu Asp Glu 50 55 <210>
SEQ ID NO 28 <211> LENGTH: 408 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-15 <400>
SEQUENCE: 28 Gly Pro Leu Gly Tyr Thr Val Tyr Asp Asp Gly Glu Glu
Lys Asn Asn 1 5 10 15 Ala Thr Val His Glu Gln Val Gly Gly Pro Ser
Leu Thr Ser Asp Leu 20 25 30 Gln Ala Gln Ser Lys Gly Asn Pro Glu
Gln Thr Pro Val Leu Lys Pro 35 40 45 Glu Glu Glu Ala Pro Ala Pro
Glu Val Gly Ala Ser Lys Pro Glu Gly 50 55 60 Ile Asp Ser Arg Pro
Glu Thr Leu His Pro Gly Arg Pro Gln Pro Pro 65 70 75 80 Ala Glu Glu
Glu Leu Cys Ser Gly Lys Pro Phe Asp Ala Phe Thr Asp 85 90 95 Leu
Lys Asn Gly Ser Leu Phe Ala Phe Arg Gly Gln Tyr Cys Tyr Glu 100 105
110 Leu Asp Glu Lys Ala Val Arg Pro Gly Tyr Pro Lys Leu Ile Arg Asp
115 120 125 Val Trp Gly Ile Glu Gly Pro Ile Asp Ala Ala Phe Thr Arg
Ile Asn 130 135 140 Cys Gln Gly Lys Thr Tyr Leu Phe Lys Gly Ser Gln
Tyr Trp Arg Phe 145 150 155 160 Glu Asp Gly Val Leu Asp Pro Asp Tyr
Pro Arg Asn Ile Ser Asp Gly 165 170 175 Phe Asp Gly Ile Pro Asp Asn
Val Asp Ala Ala Leu Ala Leu Pro Ala 180 185 190 His Ser Tyr Ser Gly
Arg Glu Arg Val Tyr Phe Phe Lys Gly Lys Gln 195 200 205 Tyr Trp Glu
Tyr Gln Phe Gln His Gln Pro Ser Gln Glu Glu Cys Glu 210 215 220 Gly
Ser Ser Leu Ser Ala Val Phe Glu His Phe Ala Met Met Gln Arg 225 230
235 240 Asp Ser Trp Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr
Ser 245 250 255 Ala Gly Thr Arg Gln Pro Gln Phe Ile Ser Arg Asp Trp
His Gly Val 260 265 270 Pro Gly Gln Val Asp Ala Ala Met Ala Gly Arg
Ile Tyr Ile Ser Gly 275 280 285 Met Ala Pro Arg Pro Ser Leu Ala Lys
Lys Gln Arg Phe Arg His Arg 290 295 300 Asn Arg Lys Gly Tyr Arg Ser
Gln Arg Gly His Ser Arg Gly Arg Asn 305 310 315 320 Gln Asn Ser Arg
Arg Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser 325 330 335 Ser Glu
Glu Ser Asn Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met 340 345 350
Asp Trp Leu Val Pro Ala Thr Cys Glu Pro Ile Gln Ser Val Phe Phe 355
360 365 Phe Ser Gly Asp Lys Tyr Tyr Arg Val Asn Leu Arg Thr Arg Arg
Val
370 375 380 Asp Thr Val Asp Pro Pro Tyr Pro Arg Ser Ile Ala Gln Tyr
Trp Leu 385 390 395 400 Gly Cys Pro Ala Pro Gly His Leu 405
<210> SEQ ID NO 29 <211> LENGTH: 195 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide RCP-16 <400>
SEQUENCE: 29 Gly Pro Leu Gly Pro Ser Gln Glu Glu Cys Glu Gly Ser
Ser Leu Ser 1 5 10 15 Ala Val Phe Glu His Phe Ala Met Met Gln Arg
Asp Ser Trp Glu Asp 20 25 30 Ile Phe Glu Leu Leu Phe Trp Gly Arg
Thr Ser Ala Gly Thr Arg Gln 35 40 45 Pro Gln Phe Ile Ser Arg Asp
Trp His Gly Val Pro Gly Gln Val Asp 50 55 60 Ala Ala Met Ala Gly
Arg Ile Tyr Ile Ser Gly Met Ala Pro Arg Pro 65 70 75 80 Ser Leu Ala
Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr 85 90 95 Arg
Ser Gln Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser Arg Arg 100 105
110 Pro Ser Arg Ala Thr Trp Leu Ser Leu Phe Ser Ser Glu Glu Ser Asn
115 120 125 Leu Gly Ala Asn Asn Tyr Asp Asp Tyr Arg Met Asp Trp Leu
Val Pro 130 135 140 Ala Thr Cys Glu Pro Ile Gln Ser Val Phe Phe Phe
Ser Gly Asp Lys 145 150 155 160 Tyr Tyr Arg Val Asn Leu Arg Thr Arg
Arg Val Asp Thr Val Asp Pro 165 170 175 Pro Tyr Pro Arg Ser Ile Ala
Gln Tyr Trp Leu Gly Cys Pro Ala Pro 180 185 190 Gly His Leu 195
<210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 30 Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe
Asn Val Asp 1 5 10 15 Lys Lys Cys Gln 20 <210> SEQ ID NO 31
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 31 Lys Gly Arg
Cys Thr Glu Gly Phe Asn Val Asp Lys Lys Cys Gln Cys 1 5 10 15 Asp
Glu Leu Cys 20 <210> SEQ ID NO 32 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 32 Glu Gly Phe Asn Val Asp Lys Lys
Cys Gln Cys Asp Glu Leu Cys Ser 1 5 10 15 Tyr Tyr Gln Ser 20
<210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 33 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr
Gln Ser Cys 1 5 10 15 Cys Thr Asp Tyr 20 <210> SEQ ID NO 34
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 34 Cys Cys Thr
Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly 1 5 10 15 Asp
Val Phe Thr 20 <210> SEQ ID NO 35 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide <400> SEQUENCE: 35 Thr Ala Glu Cys Lys Pro Gln Val
Thr Arg Gly Asp Val Phe Thr Met 1 5 10 15 Pro Glu Asp Glu 20
<210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Peptide <400>
SEQUENCE: 36 Cys Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val
Thr Arg Gly 1 5 10 15 Glu Val Phe Thr 20 <210> SEQ ID NO 37
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide <400> SEQUENCE: 37 Thr Ala Glu
Cys Lys Pro Gln Val Thr Arg Gly Glu Val Phe Thr Met 1 5 10 15 Pro
Glu Asp Glu 20 <210> SEQ ID NO 38 <211> LENGTH: 142
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Peptide RCP-17aa <400> SEQUENCE: 38 Asp Gln Glu Ser Cys Lys
Gly Arg Cys Thr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln
Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp
Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val 35 40 45
Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu Ser Glu Gly Ser Glu 50
55 60 Asp Ile Phe Glu Leu Leu Phe Trp Gly Arg Thr Ser Ala Gly Thr
Arg 65 70 75 80 Gln Pro Gln Phe Ile Ser Arg Asp Trp His Gly Val Pro
Gly Gln Val 85 90 95 Asp Ala Ala Met Ala Gly Arg Ile Tyr Ile Ser
Gly Met Ala Pro Arg 100 105 110 Pro Ser Leu Ala Lys Lys Gln Arg Phe
Arg His Arg Asn Arg Lys Gly 115 120 125 Tyr Arg Ser Gln Arg Gly His
Ser Arg Gly Arg Asn Gln Asn 130 135 140 <210> SEQ ID NO 39
<211> LENGTH: 143 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Peptide RCP-17 <400> SEQUENCE: 39 Met
Asp Gln Glu Ser Cys Lys Gly Arg Cys Thr Glu Gly Phe Asn Val 1 5 10
15 Asp Lys Lys Cys Gln Cys Asp Glu Leu Cys Ser Tyr Tyr Gln Ser Cys
20 25 30 Cys Thr Asp Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg
Gly Asp 35 40 45 Val Phe Thr Met Pro Glu Asp Glu Ser Gln Glu Glu
Ser Glu Gly Ser 50 55 60 Glu Asp Ile Phe Glu Leu Leu Phe Trp Gly
Arg Thr Ser Ala Gly Thr 65 70 75 80 Arg Gln Pro Gln Phe Ile Ser Arg
Asp Trp His Gly Val Pro Gly Gln 85 90 95 Val Asp Ala Ala Met Ala
Gly Arg Ile Tyr Ile Ser Gly Met Ala Pro
100 105 110 Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg His Arg Asn
Arg Lys 115 120 125 Gly Tyr Arg Ser Gln Arg Gly His Ser Arg Gly Arg
Asn Gln Asn 130 135 140
* * * * *