U.S. patent application number 10/384743 was filed with the patent office on 2003-08-28 for method of screening tgf-beta-inhibiting substances.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. Invention is credited to Ohtomo, Toshihiko, Ono, Koichiro, Tsuchiya, Masayuki.
Application Number | 20030162228 10/384743 |
Document ID | / |
Family ID | 17752897 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162228 |
Kind Code |
A1 |
Ono, Koichiro ; et
al. |
August 28, 2003 |
Method of screening TGF-beta-inhibiting substances
Abstract
A method for screening substances that inhibit binding between a
TAK1 polypeptide and a TAB1 polypeptide, which comprises contacting
the TAB1 polypeptide to the TAK1 polypeptide and a test sample and
then detecting or determining the TAK1 polypeptide that is bound to
the TAB1 polypeptide.
Inventors: |
Ono, Koichiro; (Gotenba-shi,
JP) ; Ohtomo, Toshihiko; (Gotenba-shi, JP) ;
Tsuchiya, Masayuki; (Gotenba-shi, JP) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
|
Family ID: |
17752897 |
Appl. No.: |
10/384743 |
Filed: |
March 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10384743 |
Mar 11, 2003 |
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10158895 |
Jun 3, 2002 |
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6551840 |
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10158895 |
Jun 3, 2002 |
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09529279 |
Apr 11, 2000 |
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6451617 |
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09529279 |
Apr 11, 2000 |
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PCT/JP98/04796 |
Oct 22, 1998 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 2333/495 20130101;
G01N 33/68 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 22, 1997 |
JP |
9-290188 |
Claims
1. A method for screening substances that inhibit binding between a
TAK1 polypeptide and a TAB1 polypeptide, which method comprises
contacting a TAB1 polypeptide to a TAK1 polypeptide and a test
sample, and then detecting or determining the TAK1 polypeptide that
is bound to the TAB1 polypeptide.
2. A method for screening substances that inhibit binding between
the TAK1 polypeptide and the TAB1 polypeptide, which method
comprises contacting a TAK1 polypeptide to a TAB1 polypeptide and a
test sample, and then detecting or determining the TAB1 polypeptide
that is bound to the TAK1 polypeptide.
3. The screening method according to claim 1 or 2, wherein the TAB1
polypeptide has an amino acid sequence comprising Met at amino acid
position 1 to Pro at amino acid position 504 of the amino acid
sequence as set forth in SEQ ID NO: 2 or having an amino acid
sequence modified by the substitution, deletion and/or addition of
one or a plurality of amino acid residues of the amino acid
sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide.
4. The screening method according to claim 1 or 2, wherein the TAK1
polypeptide has an amino acid sequence comprising Met at amino acid
position 1 to Ser at amino acid position 579 of the amino acid
sequence as set forth in SEQ ID NO: 4 or having an amino acid
sequence modified by the substitution, deletion and/or addition of
one or a plurality of amino acid residues of the amino acid
sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
5. The screening method according to any of claims 1 to 4, wherein
the TAB1 polypeptide is fused to another peptide or
polypeptide.
6. The screening method according to any of claims 1 to 4, wherein
the TAK1 polypeptide is fused to another peptide or
polypeptide.
7. The screening method according to any of claims 3 to 6, wherein
the TABI polypeptide is bound to a support.
8. The screening method according to any of claims 2 to 6, wherein
the TAK1 polypeptide is bound to a support.
9. The screening method according to claim 7 or 8 wherein the
support is beads or a plate.
10. The screening method according to claims 1 to 6 wherein the
TAK1 polypeptide, the TAB1 polypeptide, and the test sample are
contacted under a homogeneous condition.
11. The screening method according to any of claim 1 and claims 3
to 10, wherein the TAK1 polypeptide is a labeled TAK1 polypeptide
and said label is detected or determined.
12. The screening method according to any of claims 2 to 10,
wherein the TAB1 polypeptide is a labeled TAB1 polypeptide and said
label is detected or determined.
13. The screening method according to claim 11 or 12 wherein the
labeled TAK1 polypeptide or the labeled TAB1 polypeptide is labeled
with a radioisotope, an enzyme or a fluorescent substance.
14. The screening method according to any of claims 3 to 10,
wherein the TAK1 polypeptide that is bound to the TAB1 polypeptide
is detected or determined by a primary antibody against the TAK1
polypeptide or a primary antibody against another peptide or
polypeptide that is fused to the TAK1 polypeptide.
15. The screening method according to any of claims 2 to 10,
wherein the TAK1 polypeptide that is bound to the TAB1 polypeptide
is detected or determined by a primary antibody against the TAK1
polypeptide or a primary antibody against another peptide or
polypeptide that is fused to the TAK1 polypeptide, and a secondary
antibody against the primary antibody.
16. The screening method according to any of claims 3 to 10,
wherein the TAB1 polypeptide that is bound to the TAK1 polypeptide
is detected or determined by a primary antibody against the TAB1
polypeptide or a primary antibody against another peptide or
polypeptide, that is fused to the TAB1 polypeptide.
17. The screening method according to any of claims 2 to 10,
wherein the TAB1 polypeptide that is bound to the TAK1 polypeptide
is detected or determined by a primary antibody against the TAB1
polypeptide or a primary antibody against another peptide or
polypeptide that is fused to the TAB1 polypeptide, and a secondary
antibody against the primary antibody.
18. The screening method according to any of claims 14 to 17,
wherein the primary antibody or secondary antibody is labeled with
a radioisotope, an enzyme or a fluorescent substance.
19. A substance obtained by the screening method according to any
of claims 1 to 18.
20. A substance that inhibits binding between a TAK1 polypeptide
and a TAB1 polypeptide obtained by a screening method according to
any of claims 1 to 18.
21. An inhibitor of signal transduction of TGF-.beta. comprising a
substance that inhibits binding between a TAK1 polypeptide and a
TAB1 polypeptide obtained by a screening method according to any of
claims 1 to 18.
22. An activator of signal transduction of TGF-.beta. comprising a
substance that inhibits binding between a TAK1 polypeptide and a
TAB1 polypeptide obtained by a screening method according to any of
claims 1 to 18.
23. A suppressor of the enhanced production of extracellular matrix
protein comprising as. an active ingredient a substance that
inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide
obtained by a screening method according to any of claims 1 to
18.
24. An activator of the enhanced production of extracellular matrix
protein comprising as an active ingredient a substance that
inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide
obtained by a screening method according to any of claims 1 to
18.
25. A suppressor of the inhibition of cellular growth comprising as
an active ingredient a substance that inhibits binding between a
TAK1 polypeptide and a TAB1 polypeptide obtained by a screening
method according to any of claims 1 to 18.
26. An activator of the inhibition of cellular growth comprising as
an active ingredient a substance that inhibits binding between a
TAK1 polypeptide and a TAB1 polypeptide obtained by a screening
method according to any of claims 1 to 18.
27. A suppressor of monocyte migration comprising as an active
ingredient a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide obtained by a screening method
according to any of claims 1 to 18.
28. An activator of monocyte migration comprising as an active
ingredient a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide obtained by a screening method
according to any of claims 1 to 8.
29. A suppressor of the induction of biologically active substances
comprising as an active ingredient a substance that inhibits
binding between a TAK1 polypeptide and a TAB1 polypeptide obtained
by the screening method according to any of claims 1 to 18.
30. An activator of the induction of biologically active substances
comprising as an active ingredient a substance that inhibits
binding between a TAK1 polypeptide and a TAB1 polypeptide obtained
by a screening method according to any of claims 1 to 18.
31. A suppressor of immunosuppressive action comprising as an
active ingredient a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide obtained by a screening method
according to any of claims 1 to 18.
32. An activator of immunosuppressive action comprising as an
active ingredient a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide obtained by a screening method
according to any of claims 1 to 18.
33. A suppressor of the deposition of amyloid .beta. protein
comprising as an active ingredient a substance that inhibits
binding between a TAK1 polypeptide and a TAB1 polypeptide obtained
by a screening method according to any of claims 1 to 18.
34. An activator of the deposition of amyloid .beta. protein
comprising as an active ingredient a substance that inhibits
binding between a TAK1 polypeptide and a TAB1 polypeptide obtained
by a screening method according to any of claims 1 to 18.
35. A substance that inhibits the function of a TAK1 polypeptide
obtained by a screening method according to any of claims 1 to
18.
36. The substance according to claim 35 in which the function is a
kinase activity.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for screening
substances that inhibit binding between TAK1 and TAB1. The present
invention also relates to a method for screening substances that
inhibit the signal transduction of transforming growth
factor-.beta. (TGF-.beta.). The present invention further relates
to substances and uses thereof obtainable by the method for
screening substances that inhibit binding between TAK1 and
TAB1.
BACKGROUND ART
[0002] Transforming growth factor.beta. (TGF-.beta.) is a
multilfunctional factor that controls various aspects of cell
functions. As one such function, TGF-.beta. is responsible for the
repair and regeneration of tissues associated with various injuries
(Border, W. A. & Noble, N. A., The New England Journal of
Medicine (1994) 331, 1286-1292).
[0003] An abnormal production of TGF-.beta. in chronic injuries can
sometimes disturb balances in the repair and regeneration of
tissues resulting in pathological fibrosis. As a pathological
condition in which the balance of TGF-.beta. production has been
disturbed, hepatic fibrosis is known. It has been elucidated that
TGF-.beta. acts as a main causative agent of fibrosis of various
organs such as the liver, by enhancing the production of
extracellular matrix protein that can cause fibrosis, inhibiting
the synthesis of proteolytic enzymes of extracellular matrix, and
by inducing substances that inhibit proteolytic enzymes of
extracellular matrix (Border, W. A. & Noble, N. A. , The New
England Journal of Medicine (1994) 331, 1286-1292).
[0004] Other known functions of TGF-.beta. include the activity. of
inhibiting cellular growth (Moses, H. L. et al., Cell (1990) 63,
245-247), the activity of migrating monocytes (Wahl, S. M. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity
of inducing biologically active substances (Wahl, S. M. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity
of facilitating the deposition of amyloid .beta. protein
(Wyss-Coray, T. et al., Nature (1997) 389, 603-606), and the
like.
[0005] TGF-.beta. transduces its signals through heteromer
complexes of type I and type II TGF-.beta. receptors and
transmembrane proteins containing the serine- and
threonine-specific kinase domains at the side of cytoplasm (Wrana,
J. L. et al., Nature (1994) 370, 341; Kingsley, D. M. et al., Genes
Dev. (1994) 8, 133). However, much of the mechanism of signaling
downward from the TGF-.beta. receptor into the cell on the
molecular level remains to be elucidated.
[0006] As a series of systems involved in the signal transduction
of the TGF-.beta. superfamily, mitogen-activated protein kinase
(MAPK) is known.
[0007] The MAPK system is a conserved eukaryotic signaling system
that converts signals of a receptor into various functions. The
MAPK system contains three types of protein kinases, i.e.
mitogen-activated protein kinase kinase kinase (MAPKKK),
mitogen-activated protein kinase kinase (MAPKK), and
mitogen-activated protein kinase (MAPK). MAPK is activated through
phosphorylation by MAPKK. MAPKK is activated through
phosphorylation by MAPKKK (Nishida, E. et al., Trends Biochem. Sci.
(1993) 18, 128; Blumer, K. J. et al., Trends Biochem. Sci. (1993)
19, 236; David R. J. et al., Trends Biochem. Sci. (1993) 19, 470;
Marchall, C. J. et al., Cell (1995) 80, 179).
[0008] TAK1 (TGF-.beta.-activated kinase 1), that is a member of
the MAPKKK family that functions in the signaling system of
biologically active substances and that belongs to the TGF-.beta.
superfamily, was identified by Yamaguchi, K. et al. (Yamaguchi, K.
et al., Science (1995) 270, 2008).
[0009] TAB1 (TAK1 binding protein 1), a protein involved in the
signaling system of TGF-.beta. that binds to and activates TAK1,
was identified by Shibuya, H. et al. (Shibuya, H. et al., Science
(1996) 272, 1179-1182).
[0010] Although TAB1 transduces the signal of TGF-.beta. by binding
to TAK1 and activating TAK1 kinase activity, no attempts have been
made so far to search for substances that inhibit binding between
TAK1 and TAB1 in order to suppress or activate signal transduction
of TGF-.beta. by focusing on the binding between TAK1 and TAB1.
DISCLOSURE OF THE INVENTION
[0011] The present invention is intended to provide a method for
screening substances that inhibit binding between TAK1 and TAB1.
The present invention is also intended to provide a method for
screening substances that suppress or activate the signal
transduction of TGF-.beta.. The present invention further is
intended to provide substances that are obtainable by a method for
screening substances that inhibit binding between TAK1 and
TAB1.
[0012] Thus, the present invention provides (1) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide to the TAK1 polypeptide and a test
sample and then detecting or determining the TAK1 polypeptide that
is bound to the TAB1 polypeptide. Preferably, the TAB1 polypeptide
is a TAB1 polypeptide that has been bound to a support. A preferred
support is beads or a plate. In another preferred embodiment, the
contact between a TAK1 polypeptide, a TAB1 polypeptide and a test
sample is carried out in a homogeneous system.
[0013] The present invention also provides (2) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide to the TAB1 polypeptide and a test
sample, and then detecting or determining the TAB1 polypeptide that
is bound to the TAK1 polypeptide. Preferably, the TAK1 polypeptide
is a TAK1 polypeptide that has been bound to a support. A preferred
support is beads or a plate. In another preferred embodiment, the
contact between a TAK1 polypeptide, a TAB1 polypeptide and a test
sample is carried out in a homogeneous system.
[0014] The present invention also provides (3) a screening method
described in the above (1) and (2), which method comprises using a
TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2, or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide; and/or
[0015] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4, or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0016] The present invention also provides (4) a screening method
described in the above (1) to (3), which comprises using a TAK1
polypeptide fused to another peptide or polypeptide and/or a TAB1
polypeptide fused to another peptide or polypeptide.
[0017] The present invention also provides (5) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide to the labeled TAK1 polypeptide and
a test sample, and then detecting or determining the labeled TAK1
polypeptide that is bound to the TAB1 polypeptide. Preferably, the
TAB1 polypeptide is a TAB1 polypeptide that has been bound to a
support. A preferred support is beads or a plate. In another
preferred embodiment, the contact between a TAK1 polypeptide, a
TAB1 polypeptide and a test sample is carried out in a homogeneous
system.
[0018] The present invention also provides (6) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide to the labeled TAB1 polypeptide and
a test sample, and then detecting or determining the labeled TAB1
polypeptide that is bound to the TAK1 polypeptide. Preferably, the
TAK1 polypeptide is a TAK1 polypeptide that has been bound to a
support. A preferred support is beads or a plate. In another
preferred embodiment, the contact between a TAK1 polypeptide, a
TAB1 polypeptide and a test sample is carried out in a homogeneous
system.
[0019] The present invention also provides (7) a screening method
described in the above (5) and (6), which method comprises using a
TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2 or having an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide; and/or
[0020] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4 or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0021] The present invention also provides (8) a screening method
described in the above (5) to (7), which method comprises using a
TAK1 polypeptide fused to another peptide or polypeptide and/or a
TAB1 polypeptide fused to another peptide or polypeptide.
Preferably, said labeled TAK1 polypeptide or said labeled TAB1
polypeptide is a TAK1 polypeptide or a TAB1 polypeptide that is
labeled with a radioisotope, an enzyme or a fluorescent
substance.
[0022] The present invention also provides (9) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide to the TAK1 polypeptide and a test
sample, and then detecting or determining the TAK1 polypeptide that
is bound to the TAB1 polypeptide by a primary antibody against the
TAK1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1
polypeptide that has been bound to a support. A preferred support
is beads or a plate. Preferably, the primary antibody is a primary
antibody that is labeled with a radioisotope or an enzyme. In
another preferred embodiment, the contact between a TAK1
polypeptide, a TAB1 polypeptide and a test sample is carried out in
a homogeneous system.
[0023] The present invention also provides (10) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide to the TAB1 polypeptide and a test
sample, and then detecting or determining the TAB1 polypeptide that
is bound to the TAK1 polypeptide by a primary antibody against the
TAB1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1
polypeptide that has been bound to a support. A preferred support
is beads or a plate. Preferably, the primary antibody is a primary
antibody that is labeled with a radioisotope, an enzyme or a
fluorescent substance. In another preferred embodiment, the contact
between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is
carried out in a homogeneous system.
[0024] The present invention also provides (11) a screening method
described in the above (9) and (10), which method comprises using a
TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2 or having an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide.
[0025] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4 or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0026] The present invention also provides (12) a screening method
described in the above (9) to (11), which method comprises using a
TAK1 polypeptide fused to another peptide or polypeptide and/or a
TAB1 polypeptide fused to another peptide or polypeptide.
[0027] The present invention also provides (13) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide or the TAB1 polypeptide fused to
another peptide or polypeptide to the TAK1 polypeptide fused to
another peptide or polypeptide and a test sample, and then
detecting or determining the TAK1 polypeptide fused to another
peptide or polypeptide said TAK1 polypeptide being bound to the
TAB1 polypeptide or the TAB1 polypeptide fused to another peptide
or polypeptide by a primary antibody against the other peptide or
polypeptide. Preferably, the TAB1 polypeptide or the TAB1
polypeptide fused to another peptide or polypeptide is a TAB1
polypeptide or a TAB1 polypeptide fused to another peptide or
polypeptide, that has been bound to a support. A preferred support
is beads or a plate. Preferably, the primary antibody is a primary
antibody that is labeled with a radioisotope or an enzyme. In
another preferred embodiment, the contact between a TAK1
polypeptide, a TAB1 polypeptide and a test sample is carried out in
a homogeneous system.
[0028] The present invention also provides (14) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide or the TAK1 polypeptide fused to
another peptide or polypeptide to the TAB1 polypeptide fused to
another peptide or polypeptide and a test sample, and then
detecting or determining the TAB1 polypeptide fused to another
peptide or polypeptide said TAB1 polypeptide being bound to the
TAK1 polypeptide or the TAK1 polypeptide fused to another peptide
or polypeptide, by a primary antibody against the other peptide or
polypeptide. Preferably, the TAK1 polypeptide or the TAK1
polypeptide fused to another peptide or polypeptide is a TAK1
polypeptide or a TAK1 polypeptide fused to another peptide or
polypeptide, that has been bound to a support. A preferred support
is beads or a plate. Preferably, the primary antibody is a primary
antibody that is labeled with a radioisotope, an enzyme a
fluorescent substance. In another preferred embodiment, the contact
between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is
carried out in a homogeneous system.
[0029] The present invention also provides (15) a screening method
described in the above (13) and (14), which method comprises using
a TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2 or having an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide; and/or
[0030] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4 or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0031] The present invention also provides (16) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide to the TAK1 polypeptide and a test
sample, and then detecting or determining the TAK1 polypeptide that
is bound to the TAB1 polypeptide by a primary antibody against the
TAK1 polypeptide and a secondary antibody against the primary
antibody. Preferably, the TAB1 polypeptide is a TAB1 polypeptide
that has been bound to a support. A preferred support is beads or a
plate. Preferably, the secondary antibody is a secondary antibody
that is labeled with a radioisotope, an enzyme or a fluorescent
substance. In another preferred embodiment, the contact between a
TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried
out in a homogeneous system.
[0032] The present invention also provides (17) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide to the TAB1 polypeptide and a test
sample, and then detecting or determining the TAB1 polypeptide that
is bound to the TAK1 polypeptide by a primary antibody against the
TAB1 polypeptide and a secondary antibody against the primary
antibody. Preferably, the TAK1 polypeptide is a TAK1 polypeptide
that has been bound to a support. A preferred support is beads or a
plate. Preferably, the secondary antibody is a secondary antibody
that is labeled with a radioisotope, an enzyme or a fluorescent
substance. In another preferred embodiment, the contact between a
TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried
out in a homogeneous system.
[0033] The present invention also provides (18) a screening method
described in the above (16) and (17), which method comprises using
a TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2 or having an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide; and/or
[0034] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4 or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0035] The present invention also provides (19) a screening method
described in the above (16) to (18), which method comprises using a
TAK1 polypeptide fused to another peptide or polypeptide and/or a
TAB1 polypeptide fused to another peptide or polypeptide.
[0036] The present invention also provides (20) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAB1 polypeptide or the TAB1 polypeptide fused to
another peptide or polypeptide to the TAK1 polypeptide fused to
another peptide or polypeptide and a test sample, and then
detecting or determining the TAK1 polypeptide fused to another
peptide or polypeptide said TAK1 polypeptide being bound to the
TAB1 polypeptide or the TAB1 polypeptide fused to another peptide
or polypeptide, by a primary antibody against the other peptide or
polypeptide and a secondary antibody against the primary antibody.
Preferably, the TAB1 polypeptide or the TAB1 polypeptide fused to
another peptide or polypeptide is a TAB1 polypeptide or the TAB1
polypeptide fused to another peptide or polypeptide, that has been
bound to a support. A preferred support is beads or a plate.
Preferably, the secondary antibody is a primary antibody that is
labeled with a radioisotope, an enzyme or a fluorescent substance.
In another preferred embodiment, the contact between a TAK1
polypeptide, a TAB1 polypeptide and a test sample is carried out in
a homogeneous system.
[0037] The present invention also provides (21) a method for
screening substances that inhibit binding between a TAK1
polypeptide and a TAB1 polypeptide, which method comprises
contacting the TAK1 polypeptide or the TAK1 polypeptide fused to
another peptide or polypeptide to the TAB1 polypeptide fused to
another peptide or polypeptide and a test sample, and then
detecting or determining the TAB1 polypeptide fused to another
peptide or polypeptide said TAB1 polypeptide being bound to the
TAK1 polypeptide or the TAK1 polypeptide fused to another peptide
or polypeptide, by a primary antibody against the other peptide or
polypeptide and a secondary antibody against the primary antibody.
Preferably, the TAK1 polypeptide or the TAK1 polypeptide fused to
another peptide or polypeptide is a TAK1 polypeptide or the TAK1
polypeptide fused to another peptide or polypeptide, that has been
bound to a support. A preferred support is beads or a plate.
Preferably, the secondary antibody is a secondary antibody that is
labeled with a radioisotope, an enzyme a fluorescent substance. In
another preferred embodiment, the contact between a TAK1
polypeptide, a TAB1 polypeptide and a test sample is carried out in
a homogeneous system.
[0038] The present invention also provides (22) a screening method
described in the above (20) and (21), which method comprises using
a TAB1 polypeptide having an amino acid sequence comprising Met at
amino acid position 1 to Pro at amino acid position 504 of the
amino acid sequence as set forth in SEQ ID NO: 2 or having an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 2 and maintaining the
biological activity of the TAB1 polypeptide; and/or
[0039] a TAK1 polypeptide having an amino acid sequence comprising
Met at amino acid position 1 to Ser at amino acid position 579 of
the amino acid sequence as set forth in SEQ ID NO: 4 or having an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of the amino
acid sequence as set forth in SEQ ID NO: 4 and maintaining the
biological activity of the TAK1 polypeptide.
[0040] The present invention also provides a kit for conducting the
screening method as set forth in any of the above (1) to (22).
[0041] The present invention also provides a substance that is
obtainable by the screening method as set forth in any of the above
(1) to (22).
[0042] The present invention also provides a substance that
inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide
obtainable by the screening method as set forth in any of the above
(1) to (22).
[0043] The present invention also provides an inhibitor of signal
transduction of TGF-.beta., said inhibitor comprising a substance
that inhibits binding between a TAK1 polypeptide and a TAB1
polypeptide, obtainable by the screening method as set forth in any
of the above (1) to (22).
[0044] The present invention also provides an activator of signal
transduction of TGF-.beta., said activator comprising a substance
that inhibits binding between a TAK1 polypeptide and a TAB1
polypeptide, obtainable by the screening method as set forth in any
of the above (1) to (22).
[0045] The present invention also provides a suppressor of the
enhancement of extracellular matrix protein production, said
suppressor comprising, as an active ingredient, a substance that
inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide,
obtainable by the screening method as set forth in any of the above
(1) to (22).
[0046] The present invention also provides an activator of the
enhancement of extracellular matrix protein production, said
activator comprising, as an active ingredient, a substance that
inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide,
obtainable by the screening method as set forth in any of the above
(1) to (22).
[0047] The present invention also provides a suppressor of the
inhibition of cellular growth, said suppressor comprising, as an
active ingredient, a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide, obtainable by the screening
method as set forth in any of the above (1) to (22).
[0048] The present invention also provides an activator of the
inhibition of cellular growth, said activator comprising, as an
active ingredient, a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide, obtainable by the screening
method as set forth in any of the above (1) to (22).
[0049] The present invention also provides a suppressor of monocyte
migration, said suppressor comprising, as an active ingredient, a
substance that inhibits binding between a TAK1 polypeptide and a
TAB1 polypeptide, obtainable by the screening method as set forth
in any of the above (1) to (22).
[0050] The present invention also provides an activator of monocyte
migration, said activator comprising, as an active ingredient, a
substance that inhibits binding between a TAK1 polypeptide and a
TAB1 polypeptide, obtainable by the screening method as set forth
in any of the above (1) to (22).
[0051] The present invention also provides a suppressor of the
induction of a biologically active substance, said suppressor
comprising, as an active ingredient, a substance that inhibits
binding between a TAK1 polypeptide and a TAB1 polypeptide,
obtainable by the screening method as set forth in any of the above
(1) to (22).
[0052] The present invention also provides an activator of a
biologically active substance, said activator comprising, as an
active ingredient, a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide, obtainable by the screening
method as set forth in any of the above (1) to (22).
[0053] The present invention also provides a suppressor of an
immunosuppressive action, said suppressor comprising, as an active
ingredient, a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide, obtainable by the screening
method as set forth in any of the above (1) to (22).
[0054] The present invention also provides an activator of an
immunosuppressive action, said activator comprising, as an active
ingredient, a substance that inhibits binding between a TAK1
polypeptide and a TAB1 polypeptide, obtainable by the screening
method as set forth in any of the above (1) to (22).
[0055] The present invention also provides a suppressor of the
deposition of amyloid .beta. protein, said suppressor comprising,
as an active ingredient, a substance that inhibits binding between
a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the
screening method as set forth in any of the above (1) to (22).
[0056] The present invention also provides an activator of the
deposition of amyloid .beta. protein, said activator comprising, as
an active ingredient, a substance that inhibits binding between a
TAK1 polypeptide and a TAB1 polypeptide, obtainable by the
screening method as set forth in any of the above (1) to (22).
BRIEF EXPLANATION OF DRAWINGS
[0057] FIG. 1 is a diagram showing the construction of human
TAB1-FLAG and human TAK1-6xHis.
[0058] FIG. 2 is a graph showing binding between human TAK1-FLAG
and human MBP-TAB1C-FLAG.
[0059] FIG. 3 is a graph showing binding between human TAB1-FLAG
and human TAK1-6xHis.
[0060] FIG. 4 is a graph showing the activity of inhibition of
binding between human TAK1-6xHis and human MBP-TAB1C-FLAG,
determined using TAB1-FLAG as an inhibiting substance.
[0061] FIG. 5A is a graph showing the amount of fibronectin
determined in the culture supernatant of the HT/NEO cells, the
HT/DN2 cells and the HT/DN14 cells with and without the addition of
TGF-.beta.1. The values represent the mean +/- S.D. of the amount
of fibronectin in the culture supernatant prepared from three
different wells. FIG. 5B is a graph showing the amount of
fibronectin determined in the matrix extract of the HT/NEO cells,
the HT/DN2 cells and the HT/DN14 cells with and without the
addition of TGF-.beta.1. The values represent the mean +/- S.D. of
the amount of fibronectin in the matrix extract prepared from three
different wells.
[0062] FIG. 6A is a graph showing the amount of fibronectin
determined in the culture supernatant of the MES/NEO cells, the
MES/DN3 cells and the MES/DN6 cells with and without the addition
of TGF-.beta.1. The values represent the mean +/- S.D. of the
amount of fibronectin in the culture supernatant prepared from
three different wells. FIG. 6B is a graph showing the amount of
fibronectin determined in the matrix extract of the MES/NEO cells,
the MES/DN3 cells and the MES/DN6 cells with and without the
addition of TGF-.beta.1. The values represent the mean +/- S.D. of
the amount of fibronectin in the matrix extract prepared from three
different wells.
[0063] FIG. 7 is a graph showing the amount of type I collagen
determined in the culture supernatant of the MES/NEO cells, the
MES/DN3 cells and the MES/DN6 cells with and without the addition
of TGF-.beta.1. The values represent the mean +/-S.D. of the amount
of type I collagen in the culture supernatant prepared from three
different wells.
[0064] FIG. 8 is a graph showing the amount of type IV collagen
determined in the culture supernatant of the MES/NEO cells, the
MES/DN3 cells and the MES/DN6 cells with and without the addition
of TGF-.beta.1. The values represent the mean +/- S.D. of the
amount of type IV collagen in the culture supernatant prepared from
three different wells.
[0065] FIG. 9 is a graph showing the result of a two-hybrid assay
using the CHO cells. The values represent the mean +/- S.D. of the
luciferase activity in the culture supernatant prepared from three
different wells.
[0066] FIG. 10 is a graph showing the amount of PAI-1 in the
culture supernatant when TGF-.beta.1 was added to the Mv1Lu cells.
The values represent the mean +/- S.D. of the amount of PAI-1 in
the culture supernatant prepared from three different wells.
[0067] FIG. 11 is the activity in Miller Units of
.beta.-galactosidase of a yeast L40 that was transformed with an
amino terminal-truncated TAB1 mutants (TAB1C45.sup.--TAB1C20) and
the yeast 2-hybrid expression plasmid of TAK1. The measurement was
conducted three times and the result is expressed in the mean +/-
S.D. The values represent a ratio based on the .beta.-galactosidase
activity of the yeast L40 that was transformed with TAB1C68 and the
yeast 2-hybrid expression plasmid of TAK1.
[0068] FIG. 12 is the activity in Miller Units of
.beta.-galactosidase of a yeast L40 that was transformed with a
carboxy terminal-truncated TAB1 mutants (TAB1C45
.DELTA.14.sup.--TAB1C45.DELTA.25) and a yeast 2-hybrid expression
plasmid of TAK1. The measurement was conducted three times and the
result is expressed in the mean +/- S.D. The values represent a
ratio to the .beta.-galactosidase activity of the yeast L40 that
was transformed with TAB1C68 and the yeast 2-hybrid expression
plasmid of TAK1.
[0069] FIG. 13A is the result of Western analysis of TAK1 and
FLAG-TAB1 contained in the immunoprecipitate obtained using
anti-TAK1 antibody in the presence or absence of each peptide. FIG.
13B is the result obtained by quantifying the density of bands each
obtained by Western analysis and then by correcting the amount of
the co-precipitated FLAG-TAB1 with the amount of TAK1. The values
represent values relative to that obtained in the absence of the
peptide which was set as 1.
[0070] FIG. 14 shows the ability of the TAB1 deletion mutants
(TAB1C68, TAB1C45, TAB1C40, TAB1C35, TAB1C30 and TAB1C25) to bind
to and activate TAK1.
EMBODIMENT FOR CARRYING OUT THE INVENTION
[0071] The TAB1 polypeptide for use in the present invention may be
any TAB1 polypeptide, as long as it has an amino acid sequence
comprising Met at amino acid position 1 to Pro at amino acid
position 504 of the amino acid sequence as set forth in SEQ ID NO:
2 and the biological activity of the TAB1. It has been demonstrated
that the biological activity of the TAB1 polypeptide is the
activity of binding to and activating the TAK1 polypeptide.
[0072] More specifically, it has been demonstrated that the
biological activity of the TAB1 polypeptide is the activity of
binding to a region containing the catalytic domain of the TAK1
polypeptide having an amino acid sequence comprising an amino acid
Met at position 1 to an amino acid Glu at position 303 of the TAK1
polypeptide and activating the kinase activity of the TAK1
polypeptide to the MAPKK. In the present invention, however, the
TAB1 polypeptide is only required to have the activity of binding
to the TAK1 polypeptide and may be a TAB1 polypeptide that has lost
the activity of activating the TAK1 polypeptide. Accordingly, the
biological activity of the TAB1 polypeptide as used herein may be
the activity of binding to the TAK1 polypeptide.
[0073] The TAB1 polypeptide for use in the present invention may be
a TAB1 polypeptide that has the biological activity of the TAB1
polypeptide and that has an amino acid sequence modified by the
substitution, deletion and/or addition of one or a plurality of
amino acid residues of the amino acid sequence as set forth in SEQ
ID NO: 2. More specifically, the TAB1 polypeptide for use in the
present invention may have an amino acid sequence in which one or
more than one, preferably one or not greater than 20, and more
preferably one or not greater than 10 amino acid residues are
substituted in the amino acid sequence as set forth in SEQ ID NO:
2, as long as it has the biologically activity of the TAB1
polypeptide.
[0074] Alternatively, the amino acid sequence as set forth in SEQ
ID NO: 2 may be modified by deletion of one or more than one,
preferably one or not greater than 436, and more preferably one or
not greater than 10 amino acid residues. The amino acid sequence as
set forth in SEQ ID NO: 2 may also be modified by addition of one
or more than one, preferably one or not greater than 30, and more
preferably one or not greater than 20 amino acid residues. The TAB1
polypeptide for use in the present invention may also be modified
by simultaneous substitution, deletion, and/or addition of the
above amino acids.
[0075] It has been elucidated that the TAB1 polypeptide exhibits
its biological activity as long as it has an amino acid sequence
comprising amino acid Gln at position 437 to amino acid Pro at
position 504 in SEQ ID NO: 2. Thus, the TAB1 polypeptide for use in
the present invention may be a TAB1 polypeptide that has an amino
acid sequence comprising amino acid Gln at position 437 to amino
acid Pro at position 504 in SEQ ID NO: 2, or has an amino acid
sequence modified by the substitution, deletion and/or addition of
one or a plurality of amino acid residues in the amino acid
sequence comprising amino acid Met at position 1 to amino acid Asn
at position 436.
[0076] The TAB1 polypeptide may be a TAB1 polypeptide that has an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues in the amino
acid sequence comprising amino acid Gln at position 437 to amino
acid Pro at position 504 in SEQ ID NO: 2, as long as it has the
biological activity of the TAB1 polypeptide.
[0077] As a TAB1 polypeptide that has an amino acid sequence
modified by the substitution, deletion and/or addition of one or a
plurality of amino acid residues in the amino acid sequence as set
forth in SEQ ID NO: 2, there can be mentioned a TAB1 polypeptide in
which amino acid Ser at position 52 has been replaced with Arg and
a TAB1 polypeptide that has an amino acid sequence comprising amino
acid Gln at position 437 to amino acid Pro at position 504.
[0078] It is already known that a polypeptide that has an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of an amino
acid sequence retains its biological activity (Mark, D. F. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1984) 81, 5662-5666; Zoller, M. J.
& Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang,
A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1982) 79, 6409-6413).
[0079] The TAK1 polypeptide for use in the present invention may be
any TAK1 polypeptide al long as it has an amino acid sequence
comprising Met at amino acid position 1 to Ser at amino acid
position 579 in the amino acid sequence as set forth in SEQ ID NO:
4 and the biological activity of TAK1. It has been demonstrated
that the biological activity of the TAK1 polypeptide is the
activity of binding to the TAB1 polypeptide and the kinase activity
to MAPKK at an activated state.
[0080] More specifically, it has been demonstrated that it is the
activity of activating the kinase activity of MAPKK by exhibiting
the kinase activity at an activated state thereby phosphorylating
MAPKK, for example MKK3 (Moriguchi, T. et al., J. Biol. Chem.
(1996) 271, 13675-13679) and XMEK2/SEKI (Shibuya, H. et al.,
Science (1996) 272, 1179-1182). In the present invention, however,
the TAK1 polypeptide is only required to have the activity of
binding to the TAB1 polypeptide and may be a TAK1 polypeptide that
has lost the kinase activity of the TAK1 polypeptide. Accordingly,
the biological activity of the TAK1 polypeptide as used herein may
be the activity of binding to the TAB1 polypeptide.
[0081] It has been elucidated that the TAK1 polypeptide exhibits
its biological activity as long as it has an amino acid sequence
comprising amino acid Met at position 1 to amino acid Gln at
position 303 in SEQ ID NO: 4. Thus, the TAK1 polypeptide for use in
the present invention may be a TAK1 polypeptide that has an amino
acid sequence comprising amino acid Met at position 1 to amino acid
Gln at position 303 in SEQ ID NO: 4, and an amino acid sequence
modified by the substitution, deletion and/or addition of one or a
plurality of amino acid residues in the amino acid sequence
comprising amino acid Tyr at position 304 from amino acid Tyr to
amino acid Ser at position 579. The TAK1 polypeptide may be a TAK1
polypeptide that has an amino acid sequence modified by the
substitution, deletion and/or addition of one or a plurality of
amino acid residues in the amino acid sequence comprising amino
acid Met at position 1 to amino acid Gln at position 303, as long
as it has the biological activity of the TAK1 polypeptide.
[0082] The TAK1 polypeptide is activated by the binding of the TAB1
polypeptide to a region containing a catalytic domain of a TAK1
polypeptide that has an amino acid sequence comprising amino acid
Met at position 1 to amino acid Glu at position 303 of the TAK1
polypeptide as set forth in SEQ ID NO: 4. In accordance with the
present invention, it has been disclosed that the TAK1 polypeptide
binds to the TAB1 polypeptide at the amino acid sequence comprising
amino acid Val at position 76 to amino acid Gln at position 303 of
the TAK1 polypeptide as set forth in SEQ ID NO: 4. Although the
TAK1 polypeptide that has an amino acid sequence comprising amino
acid Val at position 76 to amino acid Gln at position 303 of the
TAK1 polypeptide as set forth in SEQ ID NO: 4 did not exhibit any
kinase activity, it has the activity of binding to the TAB1
polypeptide, and therefore it can be used in the present
invention.
[0083] Thus, it may be a TAK1 polypeptide that has an amino acid
sequence comprising amino acid Val at position 76 to amino acid Gln
at position 303 in SEQ ID NO: 4 and an amino acid sequence modified
by the substitution, deletion and/or addition of one or a plurality
of amino acid residues in the amino acid sequence comprising amino
acid Met at position 1 to amino acid Ile at position 75 and amino
acid Tyr at position 304.
[0084] The TAK1 polypeptide may be a TAK1 polypeptide that has an
amino acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues in the amino
acid sequence comprising amino acid Val at position 76 to amino
acid Gln at position 303 in SEQ ID NO: 4, as long as it has the
activity of binding to the TAB1 polypeptide. The biological
activity of the TAK1 polypeptide may also be activated by deleting
at least 21 amino acid residues at the amino group-side terminal
(N-terminal) of the TAK1 polypeptide.
[0085] The TAK1 polypeptide for use in the present invention may be
a TAK1 polypeptide that has the biological activity of TAK1
polypeptide and an amino acid sequence modified by the
substitution, deletion and/or addition of one or a plurality of
amino acid residues in the amino acid sequence as set forth in SEQ
ID NO: 4. More specifically, the TAK1 polypeptide for use in the
present invention may have amino acids that are substituted with
one or more than one, preferably one or not greater than 20, and
more preferably one or not greater than 10 amino acid residues in
the amino acid sequence as set forth in SEQ ID NO: 4, as long as it
has the biologically activity of the TAK1 polypeptide.
[0086] Alternatively, the amino acid sequence as set forth in SEQ
ID NO: 4 may have amino acids in which one or more than one,
preferably one or not greater than 276, and more preferably one or
not greater than 10 amino acid residues are deleted. Or, the amino
acid sequence as set forth in SEQ ID NO: 4 may have amino acids in
which one or more than one, preferably one or not greater than 30,
and more preferably one or not greater than 20 amino acid residues
are added.
[0087] As a TAK1 polypeptide that has an amino acid sequence
modified by the substitution, deletion and/or addition of one or a
plurality of amino acid residues in the amino acid sequence as set
forth in SEQ ID NO: 4, there can be mentioned a TAK1 polypeptide of
a mouse origin in which amino acid Gly at position 16 is replaced
with Ser, amino acid His at position 372 is replaced with Arg,
amino acid Ala at position 400 is replaced with Val, amino acid Thr
at position 403 is replaced with Ala, and amino acid Thr at
position 449 is replaced with Ala.
[0088] It is already known that a polypeptide that has an amino
acid sequence modified by the substitution, deletion and/or
addition of one or a plurality of amino acid residues of an amino
acid sequence retains its biological activity (Mark, D. F. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1984) 81, 5662-5666; Zoller, M. J.
& Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang,
A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1982) 79, 6409-6413).
[0089] The polypeptides for use in the present invention differ in
the amino acid sequence, molecular weight, isoelectric point, the
presence or absence of an added sugar chain, the position of an
added sugar chain, the structure of a sugar chain, the state of
phosphorylation, and/or the presence or absence of a disulfide bond
depending on the species from which they are derived, the host that
produces them, and/or the method of purification. However,
polypeptides having any structure may be used as long as they can
be suitably used in the present invention. Preferably, the species
from which the polypeptide is derived is human.
[0090] As DNA encoding the TAB1 polypeptide for use in the present
invention, there may be mentioned a nucleotide sequence comprising
base A at nucleotide position 30 to nucleotide G at position 1541
of the nucleotide sequence as set forth in SEQ ID NO: 1.
Furthermore, DNA encoding the TAB1 polypeptide for use in the
present invention can be of any origin as long as it has the base
sequence as set forth in SEQ ID NO: 1. Such DNA includes, for
example, genomic DNA, cDNA, and synthetic DNA. They may be DNA
obtained from a cDNA library and a genomic library obtained from
various cells, tissues, or organs, or from species other than
humans, and they may be a commercially available DNA library.
Vectors for use in such libraries may be plasmids, bacteriophages,
YAC vectors, and the like.
[0091] DNA encoding the TAB1 polypeptide for use in the present
invention may be DNA that hybridizes to the nucleotide sequence as
set forth in SEQ ID NO: 1 and encodes a polypeptide having the
biological activity of TAB1 . As a condition under which DNA
encoding the TAB1 polypeptide hybridizes, there may be mentioned a
stringent condition.
[0092] Such conditions include, for example, a low stringent
condition. By way of example, a low stringent condition is a
washing condition provided at room temperature in 2 .times. SSC and
0.1% sodium dodecyl sulfate. More preferably, there may be
mentioned a high stringent condition. By way of example, a high
stringent condition is a washing condition provided at 60.degree.
C. in 0.1 .times. SSC and 0.1% sodium dodecyl sulfate. It is
already known that a polypeptide encoded by a DNA that hybridizes
under a suitable condition to a base sequence encoding a
polypeptide has the same biological activity as the
polypeptide.
[0093] E. coli that has the plasmid TAB1-f-4 containing DNA
encoding the human TAB1 polypeptide having an amino acid sequence
comprising amino acid Met at amino acid position 1 to amino acid
Pro at amino acid position 504 of the amino acid sequence as set
forth in SEQ ID NO: 2 was designated as Escherichia coli DH5.alpha.
(TABI-f-4) and has been internationally deposited under the
provisions of the Budapest Treaty on Jul. 19, 1996, with the
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, of 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki, Japan, as the accession number FERM
BP-5599.
[0094] E. coli that has the plasmid pBS-TAB1 containing DNA
encoding the above human TAB1 polypeptide that comprises amino acid
Met at amino-acid position 1 to amino acid Pro at amino acid
position 504 of the amino acid sequence as set forth in SEQ ID NO:
2 and in which amino acid Ser at position 52 has been replaced with
Arg was designated as Escherichia coli HB101 (pBS-TAB1) and has
been internationally deposited under the provisions of the Budapest
Treaty on Apr. 19, 1996, with the National Institute of Bioscience
and Human Technology, Agency of Industrial Science and Technology,
of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, as the
accession number FERM BP-5508.
[0095] As DNA encoding the TAK1 polypeptide for use in the present
invention, there may be mentioned a nucleotide sequence comprising
nucleotide A at nucleotide position 183 to nucleotide G at position
1919 of the nucleotide sequence as set forth in SEQ ID NO: 2.
Furthermore, DNA encoding the TAK1 polypeptide for use in the
present invention can be of any origin as long as it has the
nucleotide sequence as set forth in SEQ ID NO: 3. Such a DNA
includes, for example, genomic DNA, cDNA, and synthetic DNA.
[0096] They may be DNA obtained from a cDNA library and a genomic
library obtained from various cells, tissues, or organs, or from
species other than human, and they may be a commercially available
DNA library. Vectors for use in such libraries may be plasmids,
bacteriophages, YAC vectors, and the like.
[0097] DNA encoding the TAK1 polypeptide for use in the present
invention may be DNA that hybridizes to the nucleotide sequence as
set forth in SEQ ID NO: 3 and encodes a polypeptide having the
biological activity of TAK1. As a condition under which the DNA
encoding the TAB1 polypeptide hybridizes, there may be mentioned a
stringent condition.
[0098] Such conditions include, for example, a low stringent
condition. By way of example, a low stringent condition is a
washing condition provided at room temperature in 2 .times. SSC and
0.1% sodium dodecyl sulfate. More preferably, there may be
mentioned a high stringent condition. By way of example, a high
stringent condition is a washing condition provided at 60.degree.
C. in 0.1 .times. SSC and 0.1% sodium dodecyl sulfate. It is
already known that a polypeptide encoded by a DNA that hybridizes
under a suitable condition to a nucleotide sequence encoding a
polypeptide has the same biological activity as the
polypeptide.
[0099] E. coli that has the plasmid pEF-TAK1 containing DNA
encoding the above mouse TAK1 polypeptide was designated as
Escherichia coli MC1061/P3 (pEF-TAK1) and has been internationally
deposited under the provisions of the Budapest Treaty on Sep. 28,
1995, with the National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology, of 1-3,
Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, as the accession
number FERM BP-5246.
[0100] E. coli that has the plasmid pEF-TAK1DN containing DNA
encoding the mouse TAK1 polypeptide that has a deletion of 21 amino
acids at the N-terminal was designated as Escherichia coli
MC1061/P3 (pEF-TAK1DN) and has been internationally deposited under
the provisions of the Budapest Treaty on Sep. 28, 1995, with the
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, of 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki, Japan, as the accession number FERM
BP-5245.
[0101] E. coli that has the plasmid phTAK1 containing DNA encoding
the human TAK1 polypeptide that has an amino acid sequence
comprising amino acid Met at amino acid position 1 to amino acid
Ser at amino acid position 579 of the amino acid sequence as set
forth in SEQ ID NO: 4 and was designated as Escherichia coli JM109
(phTAK1) and has been internationally deposited under the
provisions of the Budapest Treaty on Jul. 19, 1996, with the
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, of 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki, Japan, as the accession number FERM
BP-5598.
[0102] Polypeptides for use in the present invention may be the
above polypeptides that are fused to another peptide or
polypeptide. Such fusion polypeptides may be produced by a known
method. Another peptide or polypeptide subjected to fusion with the
polypeptide may be any peptide or polypeptide as long as it can be
advantageously used in the present invention. As such peptides, for
example, known peptides may be used including FLAG (Hopp, T. P. et
al., BioTechnology (1988) 6, 1204-1210), 6xHis comprising 6 His
(histidine) residues, 10xHis, influenza hemaglutinin (HA),
fragments of human c-myc, fragments of VSV-GP, fragments of p18HIV,
T7-tag, HSV-tag, E-tag, fragments of SV40T antigen, lck tag,
fragments of a-tubulin, B-tag, fragments of Protein C, and the
like.
[0103] As polypeptides, there may be mentioned, for example, GST
(glutathione S-transferase), HA, the constant regions of
immunoglobulin, .beta.-galactosidase, MBP (maltose-binding
protein), and the like. They may be commercially available
polypeptides.
[0104] DNA encoding the polypeptide for use in the present
invention may be generated by constructing the above-mentioned DNA
using commercially available kits or by known methods. There may be
mentioned, for example, digestion with a restriction enzyme,
addition of a linker, insertion of an initiation codon (ATG) and/or
a stop codon (ATT, TGA or TAG), and the like.
[0105] Expression vectors for use in the present invention may be
any expression vectors as long as they can be suitably used in the
present invention. As expression vectors, there may be mentioned
expression vectors. derived from a mammal such as pEF and pCDM8,
expression vectors derived from an insect such as pBacPAK8,
expression vectors derived from a plant such as pMH1 and pMH2,
expression vectors derived from an animal virus such as pHSV and
pMV, expression vectors derived from a yeast such as pNV11,
expression vectors derived from Bacillus subtilis such as pPL608
and pKTH50 and expression vectors derived from Escherichia coli
such as pGEX, pGEMEX and pMALp2.
[0106] Expression vectors of polypeptides for use in the present
invention may be produced by linking DNA encoding the TAB1
polypeptide or the TAK1 polypeptide downstream to the promoter. As
promoters/enhancers, promoters/enhancers derived from a mammal such
as the EF1-.alpha. promoter/enhancer and .gamma.-actin
promoter/enhancer, promoters/enhancers derived from an insect such
as polyhedrin virus promoter/enhancer, promoters/enhancers derived
from a plant such as tabacco mosaic virus promoter/enhancer,
promoters/enhancers derived from a plant such as SV40
promoter/enhancer and human CMV promoter/enhancer,
promoters/enhancers derived from yeast such as alcohol
dehydrogenase promoter/enhancer, promoters/enhancers derived from
Escherichia coli such as Lac promoter/enhancer, Trp
promoter/enhancer and Tac Promoter/enhancer.
[0107] For the expression of polypeptide for use in the present
invention, a signal sequence suitable for the host to be used in
the expression may be added. As the signal sequence, there may be
mentioned a signal sequence for secretary proteins. As a signal
sequence for secretary proteins, there may be mentioned a signal
sequence for secretary proteins derived from a mammal such as a
signal sequence for immunoglobulins. As a signal sequence for
secretary proteins, there may be mentioned a signal sequence for
secretary proteins derived from E. coli such as periplasm secretary
signal sequence such as OmpA and the like.
[0108] An expression vector produced as mentioned above can be
introduced into a host by a known method. Methods for introduction
into the host includes, for example, electropolation, the calcium
phosphate method, and the liposome method.
[0109] Polypeptides for use in the present invention can be
obtained as recombinant polypeptides produced using gene
recombinant technology as described above. For example, recombinant
polypeptides may be produced by cloning the base sequence of a gene
described herein from a cell, tissue, or an organ that expresses
the polypeptide and integrating the gene into a suitable vector,
which is introduced into a host to allow the host to produce said
polypeptide. The recombinant polypeptides can be used in the
present invention.
[0110] Specifically, mRNA encoding the gene can be isolated from
the cell, tissue, or organ that expresses polypeptides to be used
in the present invention. The isolation of mRNA is conducted by
preparing total RNA using a known method such as the guanidine
ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry
(1979) 18, 5294-5299), the AGPC method (Chomzynski, P. and Sacci,
N., Anal. Biochem. (1987) 162, 156-159), and then purifying mRNA
from the total RNA using the mRNA Purification Kit (Pharmacia) and
the like. Alternatively, mRNA can be prepared directly using the
QuickPrep mRNA Purification kit (Pharmacia).
[0111] The mRNA obtained is used to synthesize the cDNA of the gene
using a reverse transcriptase. The synthesis of cDNA can be
effected using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Kogyo), and the like. Alternatively, for
the synthesis and amplification of cDNA, the Marathon cDNA
Amplification kit (manufactured by CLONTECH) and the 5'-RACE method
(Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,
8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17,
2919-2932) that employs the polymerase chain reaction (PCR) may be
used.
[0112] A DNA fragment of interest may be prepared from the PCR
product thus obtained and ligated to a vector DNA. Furthermore, a
recombinant vector is constructed from this and is then introduced
into E. coli for selection of colonies to prepare the desired
recombinant vector. The nucleotide sequence of the desired DNA may
be confirmed by a known method such as the dideoxy nucleotide chain
termination method. Once the desired DNA has been obtained, it may
be integrated into an expression vector.
[0113] More specifically, the DNA constructed as described above
may be expressed to obtain polypeptides. When mammalian cells are
used, expression may be accomplished using a vector containing a
commonly used useful promoter/enhancer, the gene to be expressed,
and DNA in which the poly A signal has been operably linked at 3'
downstream thereof or a vector containing said DNA. Examples of the
promoter/enhancer include the human cytomegalovirus immediate early
promoter/enhancer.
[0114] Additionally, as the promoter/enhancer which can be used for
expression thereof, there are viral, promoters/enhancers such as
retrovirus, polyoma virus, adenovirus, and simian virus 40 (SV40),
and promoters/enhancers derived from mammalian cells such as human
elongation factor 1.alpha. (HEF1.alpha.).
[0115] For example, expression may be readily accomplished by the
method of Mulligan et al. (Nature (1979) 277, 108) when the SV40
promoter/enhancer is used, or by the method of Mizushima et al.
(Nucleic Acids Res. (1990) 18, 5322) when the HEF1.alpha.
promoter/enhancer is used.
[0116] In the case of E. coli, expression may be conducted by
operably linking a commonly used useful promoter, a signal sequence
for polypeptide secretion, and the gene to be expressed, followed
by expression thereof. As the promoter, for example, there can be
mentioned the lacz promoter and the arab promoter. The method of
Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6,
2422-2427) may be used when the lacz promoter is used, and the
method of Better et al. (Science (1988) 240, 1041-1043) may be used
when the araB promoter is used.
[0117] As a signal sequence for polypeptide secretion, when
produced in the periplasm of E. coli, the pelB signal sequence
(Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) can be
used.
[0118] As a origin of replication, there can be used those derived
from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV)
and the like. Furthermore, for the amplification of gene copy
number in the host cell system, expression vectors can include as
selectable markers the aminoglycoside transferase (APH) gene, the
thymidine kinase (TK) gene, E. coli xanthine guaninephosphoribosyl
transferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene
and the like.
[0119] For the production of polypeptides for use in the present
invention, any production system can be used. The production system
of polypeptide preparation comprises the in vitro or the in vivo
production system. As the in vitro production system, there can be
mentioned a production system which employs eukaryotic cells and
the production system which employs prokaryotic cells.
[0120] When the eukaryotic cells are used, there are the production
systems which employ the animal cells, the plant cells, and the
fungal cells. Known animal cells include (1) mammalian cells such
as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, myeloma
cells, baby hamster kidney (BHK) cells, HeLa cells, and Vero cells,
(2) amphibian cells such as xenopus oocytes (Valle, et al., Nature
(1981) 291, 358-340), or (3) insect cells such as sf9, sf21, and
Tn5. As the CHO cells, dhfr-CHO (Proc. Natl. Acad. Sci. U.S.A.
(1968) 77, 4216-4220), a CHO cell that is deficient in the DHFR
gene, and CHO K-1 (Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275)
can be preferably used.
[0121] Known plant cells include, for example, those derived from
Nicotiana tabacum, which is subjected to callus culture. Known
fungal cells include yeasts such as the genus Saccharomyces, more
specifically Saccharomyces cereviceae, or filamentous fungi such as
the genus Aspergillus, more specifically Aspergillus nicer.
[0122] When the prokaryotic cells are used, there are the
production systems which employ bacterial cells. Known bacterial
cells include Escherichia coli (E. coli), and Bacillus
subtilis.
[0123] By transforming these cells with the desired DNA and
culturing the transformed cells in vitro, polypeptides can be
obtained. Culturing is conducted in the known methods. For example,
as the culture liquid, DMEM, MEM, RPMI1640, and IMDM can be used,
and serum supplements such as fetal calf serum (FCS) may be used in
combination, or serum-free medium can be used. pH during the
culture is preferably about 6 to 8. Culture is usually carried out
at 30 to 40.degree. C. for about 15 to 200 hours with, as desired,
medium changes, aeration, and agitation.
[0124] As in vivo production systems, there can be mentioned those
which employ animals and those which employ plants. DNA of interest
is introduced into these animals or plants, and the polypeptides
are produced in such animals or plants, and recovered.
[0125] When animals are used, there are the production systems
which employ mammals and insects.
[0126] As mammals, goat, pigs, sheep, mice, and cattle can be used
(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When
mammals are used, transgenic animals can be used.
[0127] For example, a DNA of interest is inserted into the middle
of the gene encoding protein which is inherently produced in the
milk such as goat .beta. casein to prepare fusion genes. DNA
fragments containing the fusion gene into which the DNA has been
inserted are injected into a goat embryo, and the embryo is
introduced into a female goat. The polypeptide is obtained from the
milk produced by the transgenic goat born to the goat who received
the embryo or offsprings thereof. In order to increase the amount
of milk containing the polypeptide produced by the transgenic goat,
hormones may be given to the transgenic goat as appropriate.
(Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
[0128] As an insect, silkworms may be used. When silkworms are
used, baculovirus into which the DNA of interest has been inserted
is infected to the silkworm, and the desired polypeptide can be
obtained from the body fluid of the silkworm (Susumu, M. et al.,
Nature (1985) 315, 592-594).
[0129] Moreover, when plants are used, tabacco, for example, can be
used. When tabacco is used, the DNA of interest is inserted into an
expression vector for plants, for example pMON 530, and then the
vector is introduced into a bacterium such as Agrobacterium
tumefaciens. The bacterium is then infected to tabacco such as
Nicotiana tabacum to obtain the desired polypeptide from the leaves
of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994)
24, 131-138).
[0130] As methods of introducing an expression vector into a host,
there can be used a known method such as the calcium phosphate
method (virolgoy (1973) 52, 456-467), the electropolation method
(EMBO J. (1982) 1, 841-845), and the like. Considering the
frequency of use of the host's codon for use in the present
invention, a sequence having a better efficiency of expression can
be designed (Grantham, R. et al., Nucleic Acids Research (1981) 9,
r43-r74).
[0131] Gene is introduced as described above into these animals or
plants, and polypeptides are produced in the body of the animals or
the plants and recovered. Polypeptides expressed and produced as
described above can be separated from the inside or outside of the
host cell and then may be purified to homogeneity. Separation and
purification of the antibody for use in the present invention may
be accomplished by, but not limited to, the separation and the
purification methods conventionally used for protein
purification.
[0132] Polypeptides can be separated and purified by selecting and
combining, as appropriate, methods including, but not limited to,
chromatography columns such as affinity chromatography, filtration,
ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel
electrophoresis, isoelectric focusing, and the like (Antibodies: A
Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor
Laboratory, 1988).
[0133] As chromatography, there may be mentioned, for example,
affinity chromatography, ion exchange chromatography, hydrophobic
chromatography, gel-filtration, reverse phase chromatography,
adsorption chromatography, and the like (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1986). These chromatographies can be carried out using a liquid
chromatography such as HPLC and FPLC.
[0134] Polypeptides can be determined using a known method. For
example, measurement of absorbance or the Bradford method can be
used.
[0135] The present invention provides a method for screening
substances that inhibit binding between a TAK1 polypeptide and a
TAB1 polypeptide, which method comprises contacting the TAB1
polypeptide to the TAK1 polypeptide and a test sample, and then
detecting or determining the TAK1 polypeptide that is bound to the
TAB1 polypeptide; or
[0136] a method for screening substances that inhibit binding
between a TAK1 polypeptide and a TAB1 polypeptide, which method
comprises contacting the TAK1 polypeptide to the TAB1 polypeptide
and a test sample and then detecting or determining the TAB1
polypeptide that is bound to the TAK1 polypeptide.
[0137] The screening system for use in the present invention may be
conducted as an in vitro assay system.
[0138] The in vitro assay system may be conducted in a non-cellular
system. Specifically, one of the TAB1 polypeptide and the TAK1
polypeptide may be previously bound to a support, to which
polypeptide are then added the other polypeptide and the test
sample, incubated and then washed followed by detection or
determination of binding of the polypeptide to the other
polypeptide bound to the support. Alternatively, the test sample
may be added under a homogeneous condition without binding any of
the TAB1 polypeptide and the TAK1 polypeptide to the support,
incubated, and then immunoprecipitated using antibody to either of
the TAB1 polypeptide and the TAK1 polypeptide followed by detection
or determination of the amount of the conjugate.
[0139] The TAB1 polypeptide or the TAK1 polypeptide may be a
polypeptide produced by cells that inherently express them, cells
into which DNA encoding a polypeptide for use in the present
invention has been introduced, or animals or plants into which DNA
encoding a polypeptide for use in the present invention has been
introduced, which may be used in a purified or crude-purified
form.
[0140] One of the purified or semi-purified TAB1 polypeptide or the
TAK1 polypeptide is allowed to bind to the support. The polypeptide
may be immobilized onto the support by a standard method in biding
the polypeptides to a support. As supports to which polypeptides
are bound, there may be mentioned, for example, insoluble
polysaccharides such as agarose, dextran, cellulose, synthetic
resin such as polystyrene, polyacrylamide and silicone. More
specifically, commercially available beads or plates that are
produced using the above as a raw material are used. In the case of
beads, there may be used columns that are packed with them. In the
case of plates, there may be mentioned multiwell plates (96-well
multiwell plates, etc.) or biosensor chips.
[0141] Binding between polypeptides and supports may be effected
using conventionally known methods such as chemical bonding, and
physical adsorption. Alternatively, it is be possible to bind an
antibody that specifically recognizes the polypeptide to a support
in advance so that the antibody and the polypeptide become joined.
Furthermore, avidin/biotin can also be bound.
[0142] The binding between the TAB1 polypeptide and the TAK1
polypeptide may be usually effected in buffer solutions. As buffer
solutions, for example, phosphate buffers, Tris buffers and the
like may be used. Incubation conditions may be any conditions that
are conventionally used, including the incubation at 4.degree. C.
to room temperature for 1 hour to 24 hours. washing after the
incubation may be effected in any solution that does not prevent
binding between the TAB1 polypeptide and the TAK1polypeptide
including, for example, a buffer solution containing a surfactant.
As surfactants, 0.05% Tween 20 may be used.
[0143] Test specimens to be screened according to the present
invention include, for example, peptides, polypeptides, synthetic
compounds, microbial fermentation products, marine organism
extracts, plant extracts, prokaryotic cell extracts, eukaryotic
unicellular extracts, or animal cell extracts, or libraries
thereof. Substances included in these test specimens are ones that
are expected to act in an inhibitory manner on binding between the
TAK1 polypeptide and the TAB1 polypeptide. These inhibiting
substances inhibit the binding of the TAK1 polypeptide to the TAB1
polypeptide and the binding of the TAB1 polypeptide to the TAK1
polypeptide.
[0144] In order to select substances contained in these test
specimens that inhibit the binding of the TAK1 polypeptide to the
TAB1 polypeptide and the binding of the TAB1 polypeptide to the
TAK1 polypeptide, they are incubated and washed under an
appropriated condition to separate the specific binding from the
non-specific binding. Then the status of binding of the
polypeptides for use in the present invention can be evaluated.
[0145] In the screening method of the present invention, the
control group can be set up together with the group in which the
test specimens are contacted to the polypeptides. As the control
group, the negative control group having no test specimens, the
positive control group having a substance that clearly inhibits
binding between the TAB1 polypeptide and the TAK1 polypeptide, or
both of the groups can be set up.
[0146] When the bound polypeptide is detected or determined in
accordance with the present invention, the bound polypeptide can
only be detected, or the bound polypeptide may be determined in a
quantitative manner. In these cases, the result obtained for the
negative control group having no test specimens, the result
obtained for the group having a test specimen, and/or the result
obtained for the positive control group having a substance that
clearly inhibits binding between the TAB1 polypeptide and the TAK1
polypeptide can be compared to detect a substance that inhibits
binding between the TAB1 polypeptide and the TAK1 polypeptide of
interest.
[0147] Alternatively, these results may be obtained in numerical
values, which values may be compared to determine quantitatively
the activity of the substance that inhibits binding between the
TAB1 polypeptide and the TAK1 polypeptide of interest. When
quantitative determinations are made, the numerical value obtained
with the negative control group having no test specimens and those
obtained with the group in which a test specimen was applied may be
compared to detect the substance that inhibits binding between the
TAB1 polypeptide and the TAK1 polypeptide of interest. The presence
of a substance that inhibits binding between the TAB1 polypeptide
and the TAK1 polypeptide of interest in the test specimen would
decrease the bound polypeptide, thereby enabling to determine the
specimen that contains the binding-inhibiting substance.
[0148] When quantitative determinations are also made, quantitation
may be made based on a standard curve generated from the numerical
values obtained with the positive control group containing known
amounts of the substance that clearly inhibits binding between the
TAB1 polypeptide and the TAK1 polypeptide. When the amount of the
bound polypeptide is large, the activity of the substance contained
in the test specimen that inhibits binding between the TAB1
polypeptide and the TAK1 polypeptide is expected to be low, whereas
when the amount of the bound polypeptide is small, the activity of
the substance contained in the test specimen that inhibits binding
between the TAB1 polypeptide and the TAK1 polypeptide is expected
to be high.
[0149] In a screening method of a substance that inhibits binding
between the TAB1 polypeptide and the TAK1 polypeptide, biosensors
may be used that utilize a surface plasmon resonance phenomenon as
a means to detect or determine the bound polypeptide. Biosensors
that utilize a surface plasmon resonance phenomenon permit a real
time observation of protein-protein interaction as a surface
plasmon resonance signal using and without labeling a trace amount
of protein (for example BIAcore; manufactured by Pharmacia). Thus,
by using biosensors such as BIAcore, binding between the TAB1
polypeptide and the TAK1 polypeptide can be evaluated.
[0150] Accordingly, it is intended to contact the TAB1 polypeptide
or the TAK1 polypeptide to a sensor chip on which is immobilized
the TAK1 polypeptide or the TAB1 polypeptide and then to detect as
resonance signals the TAB1 polypeptide or the TAK1 polypeptide that
are bound to the TAK1 polypeptide or the TAB1 polypeptide.
[0151] Specifically it may be carried out as follows. First a
sensor chip CM5 (Biosensor) is activated and then the TAK1
polypeptide or the TAB1 polypeptide is immobilized thereon. Thus,
after the sensor chip is activated with an aqueous solution of
EDC/NHS (200 mM EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbonate,
hydrochloride), 50 mM NHS (N-hydroxysuccinimide)), it is washed
with an HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA,
0.05% Tween 20).
[0152] Then an appropriate amount of TAK1 polypeptide or TAB1
polypeptide dissolved in the HBS buffer is contacted to the sensor
chip and immobilized thereon. After washing the sensor chip with
the HBS buffer, the active groups remaining on the sensor chip are
blocked with an ethanolamine solution (1M ethanolamine
hydrochloride, pH 8.5). The sensor chip is washed again with the
HBS buffer for use in the binding evaluation.
[0153] Then an appropriate amount of TAB1 polypeptide or TAK1
polypeptide dissolved in the HBS buffer is injected, whereupon the
amount of the TAB1 polypeptide or the TAK1 polypeptide that is.
bound to the TAK1 polypeptide or the TAB1 polypeptide immobilized
on the sensor chip is observed as an increment in the value of
resonance signal.
[0154] In the above binding-evaluation system, furthermore, a test
specimen is injected after the TAB1 polypeptide or the TAK1
polypeptide. Alternatively, control groups may be set up together
with the injection of the test specimen. As the control groups, the
negative control group having no test specimens, the positive
control group having a substance that clearly inhibits binding
between the TAB1 polypeptide and the TAK1 polypeptide, or both of
the groups, can be set up.
[0155] The bound polypeptide is quantitatively determined as a
change in the value of resonance signal. In these cases, the result
obtained for the negative control group having no test specimens,
the result obtained for the group having a test specimen, and/or
the result obtained for the positive control group having a
substance that clearly inhibits binding between the TAB1
polypeptide and the TAK1 polypeptide can be compared to detect and
determine a substance that inhibits binding between the TAB1
polypeptide and the TAK1 polypeptide of interest.
[0156] As a means of detecting or determining the bound polypeptide
in the method of screening substances that inhibit binding between
the TAK1 polypeptide and the TAB1 polypeptide of the present
invention, either of the polypeptides is labeled and the label of
the bound polypeptide can be detected or determined.
[0157] For example, in the above screening method, one polypeptide
that is to be contacted to the other polypeptide together with the
test specimen is labeled beforehand and incubated with the test
specimen, washed, and then the bound polypeptide is detected or
determined by means of the label. Thus, preferably to one
polypeptide that has been bound to a support are contacted the test
specimen and the other labeled polypeptide. After incubating and
washing, the label of the bound polypeptide can be detected or
determined.
[0158] The TAK1 polypeptide or the TAB1 polypeptide can be labeled
by commonly known methods. As labels, there may be used, for
example, radioisotopes, enzymes, fluorescent substances,
biotin/avidin, and the like. These labels may be commercially
available ones. As radioisotopes, there may be mentioned, for
example, .sup.32P, .sup.33P, .sup.131I, .sup.125I, .sup.3H,
.sup.14C, and .sup.35S. As enzymes, there may be mentioned, for
example, alkaline phosphatase, horseradish peroxidase,
.beta.-galactosidase, .beta.-glucosidase, and the like. As
fluorescent substances, there may be mentioned, for example,
fluorescein isocyanate (FITC) and rhodamine. These are commercially
available and may be labeled by known methods.
[0159] Specifically, the following procedure may be used. Thus, a
solution containing one polypeptide is added to a plate, which is
then allowed to stand overnight. After washing the plate, it is
blocked with, for example, BSA to prevent non-specific binding of
polypeptides. The plate is washed again, and a test specimen and
the other polypeptide that has been labeled are added to the plate.
At the same time, a group (the negative control) containing no test
specimens and/or a group (the positive control) to which a known
concentration of a binding-inhibiting substance has been added are
set up and incubated. After incubation, the washed and bound
polypeptide is detected or determined. For the detection or
determination, in the case of a radioisotope, liquid scintillation
is used.
[0160] In the case of an enzyme, a substrate therefor is added and
the enzymatic changes, for example color development of the
substrate, are detected or determined. Comparison of these results
with the numerical value obtained for the control ,group permits
the identification of the test specimen containing the inhibiting
substance.
[0161] As a means for detecting or determining the bound
polypeptide in the method of screening substances that inhibit
binding between the TAK1 polypeptide and the TAB1 polypeptide of
the present invention, a primary antibody that specifically
recognizes one polypeptide can be used.
[0162] For example, in the above screening method, to one
polypeptide are contacted the other polypeptide together with the
test specimen, incubated with the test specimen, washed, and then
the bound polypeptide is detected or determined by means of a
primary antibody that specifically recognizes the polypeptide.
Thus, preferably to one polypeptide that has been bound to a
support are contacted the test specimen and the other polypeptide.
After incubating and washing, the bound polypeptide may be detected
or determined by means of a primary antibody that specifically
recognizes the polypeptide. Preferably, the primary antibody has
been labeled with a label. The method of producing the antibody is
described below.
[0163] The antibody can be labeled by commonly known methods. As
labels, there may be used, for example, radioisotopes, enzymes,
fluorescent substances, and the like. These labels may be
commercially available ones. As radioisotopes, there may be
mentioned, for example, .sup.32P, .sup.33P, .sup.131I, .sup.125I,
.sup.3H, .sup.14C, and .sup.35S. As enzymes, there may be
mentioned, for example, alkaline phosphatase, horseradish
peroxidase, .beta.-galactosidase, .beta.-glucosidase, and the like.
As fluorescent substances, there may be mentioned, for example,
fluorescein isocyanate (FITC) and rhodamine. These are commercially
available and may be labeled in known methods.
[0164] Specifically, the following procedure may be used. Thus, a
solution containing one polypeptide is added to a plate, which is
then allowed to stand overnight. After washing the plate, it is
blocked with, for example, BSA to prevent non-specific binding of
polypeptides. The plate is washed again, and a test specimen and
the other polypeptide are added to the plate. At the same time, a
group (the negative control) containing no test specimens and/or a
group (the positive control) to which a known concentration of a
binding-inhibiting substance has been added are set up and
incubated.
[0165] After incubation, the plate is washed and an antibody,
against the polypeptide that was added together with the test
specimen, is added. After an appropriate incubation, the plate is
washed and the polypeptide is detected or determined by means of a
primary antibody that specifically recognizes the polypeptide. For
the detection or determination, in the case of a radioisotope,
liquid scintillation is used. In the case of an enzyme, a substrate
therefor is added and the enzymatic changes, for example color
development of the substrate, are detected or determined by means
of a photometer. In the case of a fluorescent substance, detection
and determination may be effected by means of a fluorophotometer.
Comparison of these results with the numerical value obtained for
the control group permits the identification of the test specimen
containing the inhibiting substance.
[0166] As a means of detecting or determining the bound polypeptide
in the method of screening substances that inhibit binding between
the TAK1 polypeptide and the TAB1 polypeptide of the present
invention, a primary antibody that specifically recognizes the
other peptide or polypeptide fused to the TAB1 polypeptide or the
TAK1 polypeptide can be used.
[0167] For example, in the above screening method, to one
polypeptide are contacted another polypeptide together with a test
specimen, incubated with the test specimen, washed, and then the
bound polypeptide is detected or determined by means of a primary
antibody that specifically recognizes the other peptide or
polypeptide fused to the polypeptide.
[0168] Thus, preferably to one polypeptide that has been bound to a
support are contacted the test specimen and another polypeptide.
After incubating and washing, the bound polypeptide may be detected
or determined by means of a primary antibody that specifically
recognizes the other peptide or polypeptide fused to the
polypeptide. Preferably, the primary antibody has been labeled with
a label. The method of producing the antibody is described
below.
[0169] The antibody can be labeled by commonly known methods.
[0170] Specifically, the following procedure may be used. Thus, a
solution containing one polypeptide is added to a plate, which is
then allowed to stand overnight. After washing the plate, it is
blocked with, for example, BSA to prevent non-specific binding of
polypeptides. The plate is washed again, and a test specimen and a
polypeptide fused to another peptide or polypeptide are added to
the plate. At the same time, a group (the negative control)
containing no test specimens and/or a group (the positive control)
to which a known concentration of a binding-inhibiting substance
has been added are set up and incubated.
[0171] After incubation, the plate is washed and an antibody
against the other peptide or polypeptide fused to the polypeptide
that was added together with the test specimen is added. After an
appropriate incubation, the plate is washed and the polypeptide is
detected or determined by means of a primary antibody that
specifically recognizes the other polypeptide fused to the
polypeptide. For the detection or determination, in the case of a
radioisotope, liquid scintillation is used. In the case of an
enzyme, a substrate therefor is added and the enzymatic changes,
for example color development of the substrate, are detected or
determined by means of a photometer. In the case of a fluorescent
substance, detection and determination may be effected by means of
a fluorophotometer. Comparison of these results with the numerical
value obtained for the control group permits the identification of
the test specimen containing the inhibiting substance.
[0172] As a means of detecting or determining the bound polypeptide
in the method of screening substances that inhibit binding between
the TAK1 polypeptide and the TAB1 polypeptide of the present
invention, a primary antibody that specifically recognizes the TAB1
polypeptide or the TAK1 polypeptide and a secondary antibody that
specifically recognizes the primary antibody can be used.
[0173] For example, in the above screening method, to one
polypeptide are contacted another polypeptide together with a test
specimen, incubated with the test specimen, washed, and then the
bound polypeptide is detected or determined by means of a primary
antibody that specifically recognizes the polypeptide and a
secondary antibody that specifically recognizes the primary
antibody.
[0174] Thus, preferably to one polypeptide that has been bound to a
support are contacted a test specimen and another polypeptide.
After incubating and washing, the bound polypeptide may be detected
or determined by means of a primary antibody that specifically
recognizes the polypeptide and a secondary antibody that
specifically recognizes the primary antibody. Preferably, the
secondary Antibody has been labeled with a label.
[0175] The method of producing the antibody is described below.
[0176] The antibody can be labeled by commonly known methods.
[0177] Specifically, the following procedure may be used. Thus, a
solution containing one polypeptide is added to a plate, which is
then allowed to stand overnight. After washing the plate, it is
blocked with, for example, BSA to prevent non-specific binding of
polypeptides. The plate is washed again, and a test specimen and
the polypeptide are added to the plate. At the same time, a group
(the negative control) containing no test specimens and/or a group
(the positive control) to which a known concentration of a
binding-inhibiting substance has been added are set up and
incubated.
[0178] After incubation, the plate is washed and a primary antibody
against another peptide or polypeptide fused to the polypeptide
that was added together with the test specimen is added. After an
appropriate incubation, the plate is washed and the polypeptide is
detected or determined by means of the secondary antibody that
specifically recognizes the primary antibody that specifically
recognizes the polypeptide. For the detection or determination, in
the case of a radioisotope, liquid scintillation is used. In the
case of an enzyme, a substrate therefor is added and the enzymatic
changes, for example color development of the substrate, are
detected or determined by means of a photometer. In the case of a
fluorescent substance, detection and determination may be effected
by means of a fluorophotometer. Comparison of these results with
the numerical value obtained for the control group permits the
identification of the test specimen containing the inhibiting
substance.
[0179] As a means of detecting or determining the bound polypeptide
in the method of screening substances that inhibit binding between
the TAK1 polypeptide and the TAB1 polypeptide of the present
invention, a primary antibody that specifically recognizes the
other peptide or polypeptide fused to the TAB1 polypeptide or the
TAK1 polypeptide and a secondary antibody that specifically
recognizes the primary antibody can be used.
[0180] For example, in the above screening method, to one
polypeptide are contacted another polypeptide together with a test
specimen, incubated with the test specimen, washed, and then the
bound polypeptide is detected or determined by means of a primary
antibody that specifically recognizes the other peptide or
polypeptide fused to the polypeptide and a secondary antibody that
specifically recognizes the primary antibody. Thus, preferably, to
one polypeptide that has been bound to a support are contacted the
test specimen and the other polypeptide. After incubating and
washing, the bound polypeptide may be detected or determined by
means of a primary antibody that specifically recognizes the other
peptide or polypeptide fused to the polypeptide and a secondary
antibody that specifically recognizes the primary antibody.
Preferably, the secondary antibody has been labeled with a label.
The method of producing the antibody is described below.
[0181] The antibody can be labeled by commonly known methods.
[0182] Specifically, the following procedure may be used. Thus, a
solution containing one polypeptide is added to a plate, which is
then allowed to stand overnight. After washing the plate, it is
blocked with, for example, BSA to prevent non-specific binding of
polypeptides. The plate is washed again, and a test specimen and
another polypeptide fused to the other peptide or polypeptide are
added to the plate. At the same time, a group (the negative
control) containing no test specimens and/or a group (the positive
control) to which a known concentration of a binding-inhibiting
substance has been added are set up and incubated.
[0183] After incubation, the plate is washed and a primary antibody
against the other peptide or polypeptide fused to the polypeptide
that was added together with the test specimen is added. After an
appropriate incubation, the plate is washed and a secondary
antibody that specifically recognizes the primary antibody is
added. After an appropriate incubation, the plate is washed and the
polypeptide is detected or determined by means of the secondary
antibody that specifically recognizes the primary antibody that
specifically recognizes the other polypeptide fused to the
polypeptide. For the detection or determination, in the case of a
radioisotope, liquid scintillation is used. In the case of an
enzyme, a substrate therefor is added and the enzymatic changes,
for example color development of the substrate, are detected or
determined by means of a photometer. In the case of a fluorescent
substance, detection and determination may be effected by means of
a fluorophotometer. Comparison of these results with the numerical
value obtained for the control group permits the identification of
the test specimen containing the inhibiting substance.
[0184] More specifically, the present invention may be conducted
by, most specifically, an ELISA (enzyme-linked immunosorbent
assay). Thus, the TAK1 polypeptide fused to another peptide or
polypeptide, for example 6.times.His, is diluted in an
immobilization buffer (0.1 M NaHCO.sub.3, 0.02% NaN.sub.3, pH 9.6).
A suitable amount of an aqueous solution that was diluted is added
to each well of a 96-well immunoplate (manufactured by Nunc), which
is then incubated overnight at 4.degree. C.
[0185] After each well is washed three times with the wash buffer
(prepared to 0.05% Tween 20 in PBS), 200 .mu.l of a 5% solution of
BSA (manufactured by SIGMA) dissolved in PBS is added to block
overnight at 4.degree. C.
[0186] Then, each well is washed three times with the wash buffer,
and appropriate amounts of the TAB1 polypeptide fused to another
peptide or polypeptide, for example FALG, and a test specimen are
added thereto and incubated at room temperature for one hour. Each
well is washed three times with the wash buffer, and 100 .mu.l of
mouse anti-FLAG M2 antibody (manufactured by IBI) dissolved to 3
mg/ml in a dilution buffer is added to each well and incubated at
room temperature for one hour.
[0187] Each well is washed three times with a wash buffer, and 100
.mu.l of alkaline phosphatase-labeled goat anti-mouse IgG antibody
(manufactured by ZYMED) diluted 1000-fold in the dilution buffer is
added to each well and incubated at room temperature for one hour.
Each well is washed five times with the wash buffer, and 100 .mu.l
of the color development solution (substrate buffer; p-nitrophenyl
phosphate dissolved to 1 mg/ml in 50 mM NaHCO.sub.3, 10 mM
MgCl.sub.2, pH 9,8, manufactured by Sigma) is added to each well
and incubated at room temperature. Subsequently, absorbance at 405
nm is measured using a microplate reader (Model 3550, manufactured
by BIO-RAD). Comparison of these results with the numerical value
obtained for the negative control group and/or positive control
group permits the identification of the test specimen containing
the inhibiting substance.
[0188] The screening method of the present invention may also be
used for the High Throughput Screening (HTS). Specifically, steps
up to the blocking may be conducted manually, and the subsequent
reactions can be automated by robotization to realize High
Throughput Screening.
[0189] Thus, the TAK1 polypeptide fused to another peptide or
polypeptide, for example 6.times.His, is diluted in the
immobilization buffer (0.1 M NaHCO.sub.3, 0.02% NaN.sub.3, pH 9.6).
A suitable amount of the aqueous solution that was diluted to each
well of a 96-well immunoplate (manufactured by Nunc) is added and
then incubated overnight at 4.degree. C.
[0190] After each well is washed three times with the wash buffer
(prepared to 0.05% Tween 20 in PBS), 200 .mu.l of a 5% solution of
BSA (manufactured by SIGMA) dissolved in PBS is added to block
overnight at 4.degree. C.
[0191] Subsequently, an immunoplate after blocking is mounted to,
for example, the Biomek 2000 HTS system (manufactured by Beckman)
and the control program of the system is executed. At this time the
delivery of the solution to each well of the immunoplate and the
removal thereof can be carried out using the Biomek 2000 HTS system
(manufactured by Beckman) and the Multipipette 96-well simultaneous
dispenser (manufactured by Sagian) as a dispensing instrument.
Washing of each well of the immunoplate can also be carried out
using the EL404 microplate washer (Bio Tek). Measurement of
absorbance can be made using the SPECTRAmax250 plate reader
(manufactured by Molecular Devices).
[0192] The program is set so as to perform the following steps.
Thus, each well is washed three times with the wash buffer,
appropriate amounts of the test specimen and the other peptide or
polypeptide diluted in the dilution buffer (1% BSA, 0.5% Tween 20,
PBS) such as the TAB polypeptide fused to MBP (maltose-binding
protein) are added. At the same time, a group (the negative
control) containing no test specimens and a group (the positive
control) to which a known concentration of a binding-inhibiting
substance has been added are set up and incubated at room
temperature for one hour.
[0193] Each well is washed three times with the wash buffer, 100
.mu.l of rabbit anti-MBP antiserum (manufactured by New England
Biolabs) is added to each well, and incubated at room temperature
for one hour. Each well is washed three times with the wash buffer,
100 .mu.l of alkaline phosphatase-labeled goat anti-mouse IgG
antibody (manufactured by TAGO) diluted 5000-fold in the dilution
buffer is added to each well, and incubated at room temperature for
one hour.
[0194] Each well is then washed five times with the wash buffer,
100 .mu.l of the color development solution (substrate buffer;
p-nitrophenyl phosphate dissolved to 1 mg/ml in 50 mM NaHCO.sub.310
mM MgCl.sub.2, pH 9,8, manufactured by sigma) is added to each
well, and incubated at room temperature. Subsequently, absorbance
at 405 nm is measured using a microplate reader, the Biomek plate
reader (manufactured by Beckman/Molecular Devices). Comparison of
these results with the numerical value obtained for the control
group permits the identification of the test specimen containing
the inhibiting substance.
[0195] Antibodies for use in the present invention may be those
that are commercially available or that are contained in
commercially available kits, or they can be obtained as monoclonal
or polyclonal antibodies using known methods.
[0196] Monoclonal antibodies can be obtained by using the desired
sensitizing antigen, which is immunized in a conventional method
for immunization, by fusing the immune cells thus obtained with
known parent cells, and screening monoclonal antibody-producing
cells using a known screening method.
[0197] Specifically, monoclonal or polyclonal antibodies may be
generated as follows.
[0198] Though the sensitizing antigen for generation of antibodies
is not limited by the animal species from which the antibodies are
obtained, it is preferably derived from a mammal from which
peptides or polypeptides actually used in the present invention are
derived, such as humans, mice, or rats. Among them, sensitizing
antigens derived from humans are preferred. When, for example, a
human TAB1 polypeptide or human TAK1 polypeptide is used as the
sensitizing antigen, the nucleotide sequence and the amino acid
sequence thereof can be obtained using the gene sequence disclosed
in the present invention. Furthermore, when other peptides or
polypeptides that are fused with the human TAB1 polypeptide or
human TAK1 polypeptide are used as the sensitizing antigen, the
peptides and the polypeptides can be chemically synthesized or can
be obtained using genetic engineering technology.
[0199] Peptides or polypeptides that are used as the sensitizing
antigen may be full-length or fragments thereof. As fragments, for
example, C-terminal fragments or N-terminal fragments may be
mentioned.
[0200] Mammals to be immunized with the sensitizing antigen are not
specifically limited, and they are preferably selected in
consideration of their compatibility with the parent cell for use
in cell fusion. They generally include rodents, lagomorphs, and
primates.
[0201] Rodents include, for example, mice, rats, hamsters, and the
like. Lagomorphs include, for example, rabbits. Primates include,
for example, monkeys. As monkeys, catarrhines (Old-World monkeys)
such as cynomolgi (crab-eating macaque), rhesus monkeys, sacred
baboons, chimpanzees etc. are used.
[0202] Immunization of animals with a sensitizing antigen is
carried out using a known method. A general method, for example,
involves the intraperitoneal or subcutaneous administration of a
sensitizing antigen to the mammal. Specifically, a sensitizing
antigen which has been diluted and suspended in an appropriate
amount of phosphate buffered saline (PBS) or physiological saline
etc. is mixed, as desired, with an appropriate amount of a
conventional adjuvant, for example Freund's complete adjuvant.
After being emulsified, it is preferably administered to the mammal
for several times every 4 to 21 days. Alternatively a suitable
carrier may be used at the time of immunization of the sensitizing
antigen. After such immunization, the increase in the desired
antibody levels in the serum is confirmed by a conventional
method.
[0203] In order to obtain polyclonal antibodies, the blood of the
mammal that was sensitized with the antigen is removed after the
increase in the desired antibody levels in the serum has been
confirmed. Serum is separated from the blood. As polyclonal
antibodies, serum containing the polyclonal antibodies may be used,
or, as desired, the traction containing the polyclonal antibodies
may be isolated from the serum.
[0204] In order to obtain monoclonal antibodies, immune cells of
the mammal that was sensitized with the antigen are removed and are
subjected to cell fusion after the increase in the desired antibody
levels in the serum has been confirmed. At this time preferred
immune cells that are subjected to cell fusion include, in
particular, the spleen cell.
[0205] The mammalian myeloma cells as other parent cells which are
subjected to cell fusion with the above-mentioned immune cells
preferably include various known cell lines such as P3
(P3X63Ag8.653) (Kearney, J. F. et al., J. Immunol. (1979) 123:
1548-1550), P3X63Ag8.U1 (Yelton, D. E., et al., Current Topics in
Microbiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G. and
Milstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC-11
(Margulies, D. H. et al., Cell (1976) 8: 405-415), SP2/0 (Shulman,
M. et al., Nature (1978) 276: 269-270), FO (de St. Groth, S. F. and
Scheidegger, D., J. Immunol. Methods (1980) 35: 1-21), S194
(Trowbridge, I. S., J. Exp. Med. (1978) 148: 313-323), R210
(Galfre, G. et al., Nature (1979) 277: 131-133) and the like.
[0206] Cell fusion between the above immune cells and the myeloma
cells may be essentially conducted in accordance with a known
method such as is described in Milstein et al. (Kohler, G. and
Milstein, C., Methods Enzymol. (1981) 73: 3-46) and the like.
[0207] More specifically, the above cell fusion is carried out in
the conventional nutrient broth in the presence of, for example, a
cell fusion accelerator. As the cell fusion accelerator, for
example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like
may be used, and, in addition, an adjuvant such as dimethyl
sulfoxide etc. may be added as desired to enhance efficiency of the
fusion.
[0208] The preferred ratio of the immune cells and the myeloma
cells to be used is, for example, 1 to 10 times more immune cells
than the myeloma cells. Examples of culture media to be used for
the above cell fusion include RPMI1640 medium and MEM culture
medium suitable for the growth of the above myeloma cell lines, and
the conventional culture medium used for this type of cell culture,
and besides a serum supplement such as fetal calf serum (FCS) may
be added.
[0209] In cell fusion, predetermined amounts of the above immune
cells and the myeloma cells are mixed well in the above culture
liquid, to which a PEG solution previously heated to about
37.degree. C., for example a PEG solution with a mean molecular
weight of about 1000 to 6000, is added at a concentration of 30 to
60% (w/v), and mixed to obtain the desired fusion cells
(hybridomas). Then by repeating the sequential addition of a
suitable culture liquid and centrifugation to remove the
supernatant, cell fusion agents etc. which are undesirable for the
growth of the hybridoma can be removed.
[0210] Said hybridoma is selected by culturing in a conventional
selection medium, for example, the HAT culture medium (a culture
liquid containing hypoxanthine, aminopterin, and thymidine).
Culturing in said HAT culture medium is continued generally for a
period of time sufficient to effect killing of the cells other than
the desired hybridoma (non-fusion cells), generally several days to
several weeks. Then, the conventional limiting dilution method is
conducted in which the hybridomas that produce the desired antibody
are screened and cloned.
[0211] In addition to obtaining the above hybridoma by immunizing
an animal other than the human with an antigen, it is also possible
to sensitize human lymphocytes infected with EB virus with a
peptide or polypeptide, cells expressing them, or their lysates in
vitro, and to allow the resulting sensitized lymphocytes to be
fused with a human-derived myeloma cell having a permanent division
potential, for example U266, and thereby to obtain a hybridoma
producing the desired human antibody having the activity of binding
the peptide or the polypeptide (see Japanese Unexamined Patent
Publication (Kokai) No. 63(1988)-17688).
[0212] Furthermore, a transgenic animal having a repertoire of
human antibody genes is immunized with the antigen peptide or
polypeptide, cells expressing them or lysates thereof to obtain the
antibody-producing cells, which are used to obtain human antibody
against the peptide or polypeptide for use in the present invention
using hybrodomas fused to myeloma cells (see International Patent
Application WO 92-03918, WO 93-2227, WO 94-02602, WO 94-25585, WO
96-33735 and WO 96-34096).
[0213] The monoclonal antibody-producing hybridomas thus
constructed can be subcultured in the conventional culture liquid,
or can be stored for a prolonged period of time in liquid
nitrogen.
[0214] In order to obtain monoclonal antibodies from said
hybridoma, there may be employed a method in which said hybridoma
is cultured in the conventional method and the antibodies are
obtained as the culture supernatant, or a method in which the
hybridoma is administered to and grown in a mammal compatible with
said hybridoma and the antibodies are obtained as the ascites. The
former method is suitable for obtaining high-purity antibodies,
whereas the latter is suitable for a large scale production of
antibodies.
[0215] In addition to using a hybridoma to produce antibody, immune
cells that produce the desired antibody, for example the sensitized
lymphocytes that have been immortalized with an oncogene, may be
used to obtain the antibody.
[0216] A monoclonal antibody thus produced can also be obtained as
a recombinant antibody by recombinant gene technology. For example,
an antibody gene may be cloned from the hybridoma or an immune cell
such as sensitized lymphocytes that produce antibodies, and is
integrated into a suitable vector which is then introduced into a
host to produce said antibody. Recombinant antibodies may also be
used in the present invention (see, for example, Borrebaeck, C. A.
K., and Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES,
published in the United Kingdom by MACMILLAN PUBLISHERS LTD.
1990).
[0217] Antibodies for use in the present invention may be antibody
fragments or modified versions thereof as long as they have the
desired binding activity. For example, as fragments of antibody,
there may be mentioned Fab, F(ab').sub.2, Fv or single-chain Fv
(scFv) in which Fv or Fv's of the H chain and the L chain were
ligated via a suitable linker. Specifically antibodies are treated
with an enzyme such as papain or pepsin, to produce antibody
fragments, or genes encoding these antibody fragments are
constructed and then introduced into an expression vector, which is
expressed in a suitable host cell (see, for example, Co, M. S. et
al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A.
H., Methods in Enzymology (1989) 178, 476-496; Plucktrun, A. and
Skerra, A., Methods in Enzymol. (1989) 178, 497-515; Lamoyi, E.,
Methods in Enzymol. (1986) 121, 652-663; Rousseaux, J. et al.,
Methods in Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B.
W., Trends Biotechnol. (1991) 9, 132-137).
[0218] Antibodies produced and expressed as described above can be
separated from the inside or outside of the host cell and then may
be purified to homogeneity. Separation and purification of the
antibody for use in the present invention may be accomplished by,
but is not limited to, the separation and the purification methods
conventionally used for proteins.
[0219] These methods include chromatography columns such as
affinity chromatography, filtration, ultrafiltration, salting-out,
dialysis, SDS polyacrylamide gel electrophoresis, isoelectric
focusing, and the like, from which methods can be selected and
combined as appropriate for separation and purification of
antibodies (Antibodies: A Laboratory Manual, Ed Harlow and David
Lane, Cold Spring Harbor Laboratory, 1988).
[0220] As columns for use in affinity chromatography, there can be
mentioned Protein A column and Protein G column. Examples of the
carriers used in the Protein A column are Hyper D, POROS, Sepharose
F. F. (Pharmacia) and the like.
[0221] As chromatography other than the above-mentioned affinity
chromatography, there can be mentioned, for example, ion exchange
chromatography, hydrophobic chromatography, gel-filtration, reverse
phase chromatography, adsorption chromatography, and the like
(Strategies for Protein Purification and Characterization: A
Laboratory Course Manual, Ed Daniel R. Marshak et al., Cold Spring
Harbor Laboratory Press, 1986). These chromatographies can be
carried out using a liquid chromatography such as HPLC and
FPLC.
[0222] The concentration measurement and activity confirmation of
the antibody obtained as above can be made by known methods such as
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
or fluorescent antibody assay.
[0223] Substances that inhibit binding between the TAB1 polypeptide
and the TAK1 polypeptide that were obtained using the screening
method of the present invention can be obtained by screening the
test compounds such as peptides, proteins, non-peptide compounds,
synthetic compounds, microbial fermentation products, marine
organism extracts, plant extracts, cell extracts, or animal cell
extracts by screening methods. These test compounds may be novel
compounds, or existing compounds.
[0224] These binding-inhibiting substances are compounds that
inhibit binding between the TAB1 polypeptide and the TAK1
polypeptide. Compounds that were changed by addition, deletion, or
substitution of part of the structure of substances that inhibit
binding between the TAB1 polypeptide and the TAK1 polypeptide
obtained by the screening method of the present invention are
included in the substances that inhibit binding between the TAB1
polypeptide and the TAK1 polypeptide obtained by the screening
method of the present invention.
[0225] Substances that inhibit binding between the TAB1 polypeptide
and the TAK1 polypeptide obtained by the screening method of the
present invention may be substances that activate signal
transduction of TGF-.beta. or substances that suppress signal
transduction of TGF-.beta.. TGF-.beta. is known to have the effect
of enhancing extracellular matrix protein production, inhibiting
cellular growth, causing monocyte migration, inducing biologically
active substances, suppressing immunity, depositing amyloid .beta.
protein, and the like. Both of the TAB1 polypeptide and the TAK1
-polypeptide are responsible for signal transduction of TGF-.beta.
each by binding thereto. Thus, substances that inhibit binding
between the TAB1 polypeptide and the TAK1 polypeptide obtained by
the screening method of the present invention can be obtained as a
substance that activates or suppresses signal transduction of
TGF-.beta..
[0226] When substances that inhibit binding between the TAB1
polypeptide and the TAK1 polypeptide obtained by the screening
method of the present invention are used as medicaments for humans
and mammals such as mice, rats, guinea pigs, rabbits, chickens,
cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees,
they may be used in the conventional method.
[0227] For example, they may be used, as desired, orally as
capsules and microcapsules, or parenterally in the form of sterile
solutions with water or other pharmaceutically acceptable liquids
or suspensions. For example, substances that inhibit binding
between the TAB1 polypeptide and the TAK1 polypeptide are produced
in unit dosage forms required for generally accepted formulations
by mixing with pharmaceutically acceptable carriers, excipients,
vehicles, antiseptics, stabilizers, and adhesion inhibitors. The
amount of active ingredients in these formulations is designed to
provide an indicated suitable range of doses. As additives that may
be blended for tables or capsules, for example, gelatin, HSA (human
serum albumin), crystalline cellulose, alginic acid, magnesium
stearate, sucrose, and lactose may be used.
[0228] As aqueous solutions for injection, there may be mentioned,
for example, isotonic liquids such as physiological saline, glucose
and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and
sodium chloride, and they may be used in combination with suitable
solubilizing agents such as alcohols, specifically ethanol,
polyalchohols including, for example, propylene glycol and
polyethylene glycol, nonionic surfactants such as polysorbate 80TM,
HCO-50, benzyl benzoate, phosphate buffer, sodium acetate buffer,
procaine hydrochloride, benzyl alchohol, and phenol.
[0229] The dosage of substances that inhibit binding between the
TAB1 polypeptide and the TAK1 polypeptide for a human adult
(assuming the body weight of 60 kg) is, when given orally, usually
about 0.1 to 100 mg/day, preferably about 1.0 to 50 mg/day, and
more preferably about 1.0 to 20 mg/day, though this may vary
depending on the medical conditions.
[0230] When given parenterally, the dose per administration for a
human adult (assuming the body weight of 60 kg) of usually about
0.01 to 30 mg/day, preferably about 0.1 to 20 mg/day, and more
preferably about 0.1 to 10 mg/day in the case of injections is
conveniently administered via intravenous injection, though this
may vary depending on the subject organ, medical conditions, and
the method of administration. For other animals, the amount
converted in terms of the body weight of 60 kg may be
administered.
EXAMPLES
[0231] The present invention will now be explained in more details
with reference to the examples. It should be noted, however, that
the present invention is not limited to them in any way.
Example 1
Construction of a Baculovirus Transfer Vector for Recombinant Human
TAB1 and Recombinant Human TAK1
[0232] In order to express a full-length human TAB1 polypeptide and
a full-length human TAK1 polypeptide by a baculovirus expression
system, a baculovirus transfer vector was constructed. At this
time, it was designed to add a peptide tag in order to facilitate
purification and detection.
[0233] Thus, a FLAG tag comprising 8 amino acids
(Asp-Tyr-Lys-Asp-Asp-Asp-- Asp-Lys; SEQ ID NO: 5) was added to the
carboxy terminal of human TAB1. Also, a 6.times.His tag (Janknecht,
R. et al., Gene (1992) 121, 321-324) comprising 6 contiguous His
residues (His-His-His-His-His-His; SEQ ID NO: 6) was added to the
carboxy terminal of human TAK1. Each recombinant polypeptide is
expressed as a fusion polypeptide, human TAB1-FLAG or human
TAK1-6.times.His.
[0234] In order to obtain a DNA fragment encoding human TAB1-FLAG,
a PCR method was carried out with plasmid pBS-TAB1 (Shibuya, H. et
al., Science (1996) 272, 1179-1182) as a template using a sense
primer TABFS (SEQ ID NO: 7) and an antisense primer TAB1AS (SEQ ID
NO: 8) that were synthesized using a primer synthesizer.
[0235] The sense primer TABFS comprises a nucleotide sequence from
nucleotide A at position 30 to nucleotide G at position 47 of the
region encoding a full-length human TAB1 polypeptide contained in
plasmid PBS-TAB1 as set forth in SEQ ID NO: 1 or 2 after the
recognition site of the restriction enzyme EcoRI. The antisense
primer TAB1AS comprises a nucleotide sequence complementary to a
series of nucleotide sequences comprising a nucleotide sequence
encoding 5 amino acid sequence comprised of Gly-Thr-Gly-Gly-Ser
(SEQ ID NO: 9), a nucleotide sequence encoding the FLAG tag, two
stop codons, and a recognition site of the restriction enzyme XbaI,
after a nucleotide sequence from nucleotide A at position 1524 to
nucleotide G at position 1541 of the region encoding the human TAB1
polypeptide contained in the plasmid pBS-TAB1 as set forth in SEQ
ID NO: 3 or 4 (FIG. 1).
[0236] The PCR method comprised a total of 25 cycles of 94.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
2 minutes per cycle. PCR reaction products were separated and
purified by a 1% low-melting point agarose gel (manufactured by
Sigma), digested with the restriction enzymes EcoRI and XbaI, and
then were inserted into the baculovirus transfer vector pBacPAK9
(manufactured by CLONTECH).
[0237] The nucleotide sequence of the inserted DNA fragment was
determined by a DNA sequencer (Model 373A, manufactured by ABI)
thereby to confirm that the correct nucleotide sequence had bee
inserted.
[0238] The plasmid comprising a nucleotide sequence encoding human
TAB1-FLAG was designated as pBacTABF. The nucleotide sequence and
the amino acid sequence encoding human TAB1-FLAG are shown in SEQ
ID NO: 10 and 11.
[0239] In order to obtain a DNA fragment encoding human
TAK1-6.times.His, a PCR method was carried out with plasmid phTAK1
(Japanese Unexamined Patent Publication (Kokai) No. 9(1997)-163990)
as a template using a sense primer TAKS (SEQ ID NO: 12) and an
antisense primer TAKAS (SEQ ID NO: 13) that were synthesized using
a primer synthesizer.
[0240] The sense primer TAKS comprises a nucleotide sequence from
nucleotide A at position 183 to nucleotide C at position 200 of the
region encoding a human TAK1 polypeptide contained in plasmid
phTAK1 as set forth in SEQ ID NO: 3 after the recognition site of
the restriction enzyme EcoRI. The antisense primer TAKAS comprises
a nucleotide sequence complementary to a nucleotide sequence
encoding 5 amino acid sequence comprised of Gly-Thr-Gly-Gly-Ser, a
nucleotide sequence encoding the 6.times.His tag, two stop codons,
and a recognition site of the restriction enzyme XbaI, after a
nucleotide sequence from nucleotide A at position 1902 to
nucleotide A at position 1919 of the region encoding the human TAK1
polypeptide contained in plasmid phTAK1 (FIG. 1).
[0241] The PCR method comprised a total of 25 cycles of 94.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
2 minutes per cycle. PCR reaction products were separated and
purified by a 1% low-melting point agarose gel (manufactured by
Sigma), digested with the restriction enzymes EcoRI and XbaI, and
then were inserted into the baculovirus transfer vector pBacPAK9
(manufactured by CLONTECH). The nucleotide sequence of the inserted
DNA fragment was determined by a DNA sequencer (Model 373A,
manufactured by ABI) thereby to confirm that the correct nucleotide
sequence had been inserted. The plasmid comprising a nucleotide
sequence encoding human TAK1-6.times.His was designated as
pBacTAKF. The nucleotide sequence and the amino acid sequence
encoding human TAK1-6.times.His are shown in SEQ ID NO: 14 and
15.
Example 2
Expression of Recombinant Human TAB1-FLAG and Human
TAK1-6.times.His Polypeptides
Example 2-1
Construction of a Recombinant Baculovirus
[0242] A recombinant baculovirus was constructed in accordance with
the instructions by CLONTECH.
[0243] Thus, for the construction of the recombinant human
TAB1-FLAG baculovirus, 0.5 .mu.g of the transfer vector pBacTABF of
the above Example 1, 5 .mu.l of the Bsu36 I digest of baculovirus
BacPAK6 DNA (manufactured by CLONTECH), and 50 .mu.l of a 0.1 mg/ml
lipofectin solution (manufactured by CLONTECH) were diluted to a
total of 100 .mu.l in distilled water. The aqueous solution was
mixed with 1.5 ml of serum-free medium and was added with
1.times.10.sup.6 lined insect cells Sf9 (ATCC CRL 1711).
[0244] At 5 hours after the addition, 1.5 ml of the insect cell
culture medium (containing a medium supplement for the Grace insect
cells, manufactured by GIBCO BRL) supplemented with 10% fetal
bovine serum was further added, incubated at 27.degree. C. for 5
days, and then the supernatant was recovered. Using the culture
supernatant, a plaque assay was carried out according to the
instructions by the manufacturer to isolate a recombinant
baculovirus that expresses recombinant TAB1-FLAG from a single
plaque.
[0245] Using the transfer vector pBacTAKF described in the above
Working Example 1 in a similar procedure, a recombinant baculovirus
expressing the recombinant human TAK1-6.times.His was
constructed.
Example 2-2
Expression of each Recombinant Polypeptide Using a Recombinant
Baculovirus
[0246] A 100 M.O.I. amount of a recombinant baculovirus was
infected to 1.times.10.sup.9 lined insect cells Sf9, and was
incubated in 1000 ml of an insect cell culture medium supplemented
with 2% fetal bovine serum at 27.degree. C. for 5 days. Cells after
incubation were washed three times in PBS (Dulbecco PBS,
manufactured by Nissui), and were subjected to the following
purification.
Example 3
Purification of Recombinant Human TAB1-FLAG and Human
TAK1-6.times.His Polypeptide
Example 3-1
Purification of the Recombinant Human TAB1-FLAG Polypeptide
[0247] (1) Preparation of a Cleared Lysate
[0248] After the cells obtained in the above 2-2 were suspended in
the TMN buffer (20 mM Tris-HCl, 3 mM MgCl.sub.2, 150 mM NaCl, 0.1
mg/ml PMSF, 1 .mu.g/ml leupeptin, 1 .mu.g/ml aprotinin, pH 7.5) to
a density of 4.times.10.sup.7 cells/ml, the cells were treated by
an ultrasonic disrupting instrument (SONIFIER 250, manufactured by
BRANSON) until 90% of the cells were disrupted. The insoluble
material in the disrupted solution was precipitated by centrifuging
by a centrifuge (model MRX-150, manufactured by TOMY) at 14,000 rpm
for 10 minutes. The supernatant thus obtained was filtered using a
0.45 .mu.m filter (SterivexTM-HV, manufactured by MILLIOPORE), and
the filtrate was used as the cleared lysate.
[0249] (2) Purification with an Anti-FLAG M2 Affinity Gel
[0250] In order to purify the recombinant human TAB1-FLAG
polypeptide from the cleared lysate, affinity purification with
anti-FLAG antibody was carried out as follows:
[0251] Two ml of the anti-FLAG antibody M2 affinity gel
(manufactured by IBI) was equilibrated with a TBS buffer (50 mM
Tris-HCl, 150 mM NaCl, pH 7.4) in the bed support (Poly-Prep
Chromatography Column, manufactured by BIO-RAD). The above
supernatant was added to the equilibrated anti-FLAG antibody M2
affinity gel, and the human TAB1-FLAG polypeptide was bound
thereto. The column was washed with 30 ml of the TBS buffer, and
the bound polypeptide was eluted with 2 ml each of the elution
buffer (0.1 M glycine-HCl, pH 3.5) in six portions.
[0252] After the elution, the elution buffer was replaced with PBS
using the gel filtration column PD-10 (manufactured by Pharmacia),
which was used as a purified product of the recombinant human
TAB1-FLAG polypeptide. The purity of the recombinant human
TAB1-FLAG polypeptide was measured by the BCA* Protein Assay
Reagent (manufactured by PIERCE) using BSA as a standard.
Example 3-2
Purification of the Recombinant Human TAK1-6.times.His
Polypeptide
[0253] (1) Preparation of the Cleared Lysate
[0254] The cells obtained in the above 2-2 were suspended in the
sonication buffer (20 mM Tris-HCl, 100 NaCl, 0.1 mg/ml PMSF, 1
.mu.g/ml leupeptin, 1 .mu.g/ml aprotinin, pH 8.0) to a density of
4.times.10.sup.7 cells/ml, and then the cells were treated by an
ultrasonic disrupting instrument (SONIFIER 250, manufactured by
BRANSON) until 90% of the cells were disrupted. The insoluble
material in the disrupted solution was precipitated by centrifuging
by a centrifuge (model MRX-150, manufactured by TOMY) at 14000 rpm
for 10 minutes. The supernatant thus obtained was filtered using a
0.45 .mu.m filter (Sterivex.TM.-HV, manufactured by MILLIOPORE),
and the filtrate was used as the cleared lysate.
[0255] (2) Purification with TALON.TM. Metal Affinity Resin
[0256] In order to purify the recombinant human TAK1-6.times.His
polypeptide from the cleared lysate, purification with affinity
resin (TALON.TM. Metal Affinity Resin, manufactured by CLON TECH)
was carried out as follows:
[0257] Two ml of the affinity resin equilibrated with the
sonication buffer and the cleared lysate of the above Working
Example 3-2 (1) were mixed under shaking for 20 minutes, to which
the recombinant human TAK1-6.times.His polypeptide was ligated.
After removing the supernatant by centrifugation at 700 .times.g,
the affinity resin was mixed under shaking in 20 ml of the wash
buffer (10 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, pH 8.0) at
4.degree. C. for 10 minutes, followed by the removal of the
supernatant by centrifugation at 700 .times.g.
[0258] After the affinity resin was mixed under shaking in 20 ml of
the wash buffer (10 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, pH
8.0) at 4.degree. C. for 10 minutes, the resin was washed by
removing the supernatant by centrifugation at 700 .times.g. The
affinity resin after washing was suspended in 2 ml of the wash
buffer and was transferred to the bed support (Poly-Prep
Chromatography Column, manufactured by BIO-RAD), and the affinity
resin was further washed with 6 ml of the wash buffer.
[0259] The affinity resin was subjected to elution by the elution
buffer (50 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, pH 8.0) in
six portions of 2 ml each. After elution, the elution buffer was
replaced with PBS using the gel filtration column PD-10
(manufactured by Pharmacia), which was used as a purified product
of the recombinant human TAK1-6.times.His polypeptide. The purity
of the recombinant human TAK1-6.times.His polypeptide was measured
by the BCA* Protein Assay Reagent (manufactured by PIERCE) using
BSA as a standard.
Example 4
Preparation of the Recombinant MBP-TAB1C-FLAG
Example 4-1 Construction of the Expression Vector
[0260] By an expression system using Escherichia coli (E. coli), an
expression vector for expression in E. coli of a fusion polypeptide
of a polypeptide comprising 81 amino acids at the carboxy terminal
of the human TAB1 polypeptide and maltose-binding protein was
constructed.
[0261] At this time, a FLAG tag was added to the carboxy terminal
of the above fusion polypeptide in order to facilitate purification
and detection.
[0262] In order to obtain a DNA fragment encoding 81 amino acids at
the carboxy terminal of the human TAB1 and the FLAG tag, a PCR
method was carried out with plasmid pBacTABF as a template using a
sense primer TABC1 (SEQ ID NO: 16) and an antisense primer TABC3
(SEQ ID NO: 17) that were synthesized using a primer
synthesizer.
[0263] The sense primer TABC1 comprises a nucleotide sequence from
nucleotide C at position 1281 to nucleotide T at position 1307 of
the region encoding the human TAB1 polypeptide contained in plasmid
pBacTABF as set forth in SEQ ID NO: 9 after the recognition site of
the restriction enzyme XmnI.
[0264] The antisense primer TABLAS comprises a nucleotide sequence
complementary to a series of nucleotide sequences comprising a
nucleotide sequence encoding a nucleotide sequence encoding the
FLAG tag, two stop codons, and a recognition site of the
restriction enzyme HindIII, after a nucleotide sequence from
nucleotide C at position 1489 to nucleotide G at position 1518 of
the region encoding the human TAB1 polypeptide contained in the
plasmid pBacTABF as set forth in SEQ ID NO: 9.
[0265] The PCR method comprised a total of 25 cycles of 94.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
1 minute per cycle. PCR reaction products were separated and
purified by a 1% low-melting point agarose gel (manufactured by
Sigma), digested with the restriction enzymes XmnI and HindIII, and
then were inserted into a fusion polypeptide expression vector
pMAL-p2, (manufactured by New England Biolabs).
Example 4-2
Expression of the Fusion Polypeptide
[0266] A fusion polypeptide was expressed according. to the
instructions by New England Biolabs. Thus, E. coli having the
plasmid obtained as above was grown overnight and 2 ml thereof was
inoculated into 200 ml of a rich broth (10 g tryptone, 5 g yeast
extract, 5 g NaCl, 2 g glucose, 100 mg ampicillin/liter). It was
incubated under shaking at 37.degree. C. till the cell density
reached A.sub.600=1, at which time IPTG (isopropylthiogalactoside)
was added to a final concentration of 0.3 mM. It was further
incubated under shaking at 37.degree. C. for 2 hours and then was
centrifuged at 4000 .times.g for 10 minutes to harvest the
cells.
Example 4-3
Preparation of the Periplasm Fraction
[0267] The cells collected in the above Example 4-2 was resuspended
in 25 ml of 30 mM Tris, 20% sucrose, pH 8.0, and 50 .mu.l of 0.5 M
EDTA, pH 8.0, was added thereto followed by incubation under
shaking for 10 minutes. Subsequently, it was cetrifuged at 8000
.times.g to remove the supernatant and the cells were resuspended
in 25 ml of ice-cold 5 mM MgSO.sub.4 and was further incubated
under shaking in the ice. After centrifugation at 8000 .times.g,
the supernatant was recovered as the periplasm fraction.
Example 4-4
Purification with the Anti-FLAG M2 Affinity Gel
[0268] In order to purify recombinant MBP-TAB1C-FLAG polypeptide
from the periplasm fraction, affinity purification with anti-FLAG
M2 antibody was carried out in a similar manner to Example 3-1 (2)
to obtain a purified product of the recombinant MBP-TAB1C-FLAG
polypeptide.
Example 5
Construction of an ELISA System Using the Purified Product
[0269] Using the purified product obtained as above an ELISA system
was constructed to detect in vitro an interaction between the
recombinant human TAB1 polypeptide or the recombinant
MBP-TAB1C-FLAG polypeptide and the recombinant human TAK1
polypeptide. In the ELISA system it is intended to contact the
recombinant human TAB1-FLAG polypeptide or the human MBP-TAB1C-FLAG
polypeptide to a 96-well immunoplate to which the human
TAK1-6.times.His polypeptide had been previously immobilized and
thereby to detect the recombinant human TAB1-FLAG polypeptide or
the recombinant MBP-TAB1C-FLAG polypeptide using a primary and
secondary antibody.
Example 5-1
Construction of an in Vitro Binding-Evaluation System
[0270] (1) The purified product of human TAK1-6.times.His
polypeptide was diluted in the immobilization buffer (0.1 M
NaHCO.sub.3, 0.02% NaN.sub.3, pH 9.6). To each well of a 96-well
immunoplate (manufactured by Nunc) was added 100 .mu.l each of the
diluted aqueous solution (equivalent to 100 ng of the human
TAK1-6.times.His polypeptide), and the plate was incubated
overnight at 4.degree. C.
[0271] After each well was washed three times with the wash buffer
(diluted to 0.05% Tween 20 in PBS), 200 .mu.l of a 5% BSA
(manufactured by SIMGA) solution dissolved in PBS was added thereto
and was blocked overnight at 4.degree. C.
[0272] Then each well was washed three times with the wash buffer,
100 .mu.l of the human MBP-TAB1C-FLAG polypeptide diluted in the
dilution buffer (1% BSA, 0.5% Tween 20, PBS) was added thereto and
was incubated at room temperature for one hour. Then each well was
washed three times with the wash buffer, 100 .mu.l of rabbit
anti-MBP antiserum (manufactured by New England Biolabs) diluted
5000-fold in the dilution buffer was added to each well and was
incubated at room temperature for one hour. Then each well was
washed three times with the wash buffer, 100 .mu.l of alkaline
phosphatase-labeled goat anti-rabbit antibody (manufactured by
TAGO) diluted 5000-fold in the dilution buffer was added to each
well and was incubated at room temperature for one hour.
[0273] After each well was washed five times with the wash buffer,
100 .mu.l of the color development solution (the substrate buffer;
p-nitrophenyl phosphate dissolved to 1 mg/ml in 50 mM NaHCO.sub.3,
10 mM MgCl.sub.2, pH 9.8, manufactured by Sigma) was added to each
well and was incubated at room temperature, and then absorbance at
405 nm was determined using a microplate reader (Model 3550,
manufactured by BIO-RAD).
[0274] The result confirmed that the absorbance increased depending
on the concentration of the recombinant MBP-TAB1C-FLAG polypeptide.
On the other hand, there were no increases in absorbance dependent
on the concentration of the recombinant MBP-TA B1C-FLAG polypeptide
in the group in which the human TAK1-6.times.His polypeptide was
not immobilized (FIG. 2). This indicated that the contacted
recombinant MBP-TAB1C-FLAG polypeptide specifically bound to the
recombinant human TAK1-6.times.His polypeptide.
[0275] (2) The purified product of human TAK1-6.times.His
polypeptide was diluted in the immobilization buffer (0.1 M
NaHCO.sub.3, 0.02% NaN.sub.3, pH 9.6). To each well of a 96-well
immunoplate (manufactured by Nunc) was added 100 .mu.l each of the
diluted aqueous solution (equivalent to 80 ng of the human
TAK1-6.times.His polypeptide), and the plate was incubated
overnight at 4.degree. C.
[0276] After each well was washed three times with the wash buffer
(diluted to 0.05% Tween 20 in PBS), 200 .mu.l of a 5%
BSA.(manufactured by SIMGA) solution dissolved in PBS was added
thereto and was blocked overnight at 4.degree. C.
[0277] Then each well was washed three times with the wash buffer,
and 100 .mu.l of the human TAB1-FLAG polypeptide diluted in the
dilution buffer (1% BSA, 0.5% Tween 20, PBS) was added and was
incubated at room temperature for one hour. Then each well was
washed three times with the wash buffer, 100 .mu.l of mouse
anti-FLAG antibody (manufactured by IBI) diluted to 3 .mu.g/ml in
the. dilution buffer was added to each well, and was incubated at
room temperature for one hour.
[0278] Then each well was washed three times with the wash buffer,
100 .mu.l of alkaline phosphatase-labeled goat anti-mouse IgG
antibody (manufactured by ZYMED) diluted 1000-fold in the dilution
buffer was added to each well and was incubated at room temperature
for one hour. After each well was washed five times with the wash
buffer, 100 .mu.l of the color development solution (the substrate
buffer; p-nitrophenyl phosphate dissolved to 1 mg/ml in 50 mM
NaHCO.sub.3, 10 mM MgCl.sub.2, pH 9.8, manufactured by Sigma) was
added to each well and was incubated at room temperature, and then
absorbance at 405 nm was determined using a microplate reader
(Model 3550, manufactured by BIO-RAD).
[0279] The result confirmed that the absorbance increased depending
on the concentration of the recombinant human TAB1-FLAG
polypeptide. On the other hand, there were no increases in
absorbance dependent on the concentration of the human TAB1-FLAG
polypeptide in the group in which the human TAK1-6.times.His
polypeptide was not immobilized (FIG. 3).
[0280] This indicated that the recombinant human TAK1-6.times.His
polypeptide that was prepared in the baculovirus expression system
specifically bound to the human TAB1-FLAG polypeptide in vitro.
Example 5-2
A Binding-Inhibition Study Using the Recombinant Human TAB1-FLAG
Polypeptide
[0281] It was investigated whether the recombinant human TAB1-FLAG
polypeptide used as an inhibiting substance inhibits binding
between the recombinant MBP-TAB1C-FLAG polypeptide and the
recombinant TAK1 polypeptide.
[0282] In a similar manner to that in the above 5-1 (2), the
recombinant human TAK1-6.times.His polypeptide was immobilized and
was blocked. Then the recombinant human TAB1-FLAG polypeptide that
was serially diluted in the dilution buffer was added as a
binding-inhibiting substance to each well together with 16.5 .mu.l
each of the recombinant MBP-TAB1C-FLAG polypeptide and
incubated.
[0283] Thereafter, absorbance was determined as in the above, the
result of which confirmed the decrease in absorbance dependent on
the concentration of the recombinant human TAB1-FLAG polypeptide
added as a binding-inhibiting substance (FIG. 4).
[0284] The foregoing has shown that the in vitro binding-evaluation
system constructed in Example 2-1 is effective as a system for
screening substances that inhibit binding between the TAK1
polypeptide and the MBP-TAB1C-FLAG polypeptide.
Example 6
Construction of the TAK1-DN Expression Vector and Establishment of
the Recombinant
[0285] In order to demonstrate that the signal transduction of
TGF-.beta. can be inhibited by inhibiting specific binding between
the human TAK1 polypeptide and the human TAB1 polypeptide, TAK1-DN
expression vector that acts as a dominant negative inhibitor was
constructed, was introduced into various cells described below, and
the reactivity to TGF-.beta. was characterized.
[0286] TAK1-DN has an amino acid sequence comprising amino acid Glu
at position 77 to amino acid Gln at position 303 of the amino acid
sequence as set forth in SEQ ID NO: 4 which is the TAB1 binding
site of the TAK1 polypeptide. The gene fragment encoding TAK1-DN
was amplified using ph-TAK1 (Japanese Unexamined Patent Publication
(Kokai) No. 9(1997.)-163990) by the PCR method. Thus, using a sense
primer TAK1S (SEQ ID NO: 18) containing the restriction enzyme
EcoRI recognition site and the initiation codon ATG and an
antisense primer TAK1S (SEQ ID NO: 19) containing the restriction
enzyme NotI recognition site and the stop codon, a DNA fragment
encoding TAK1-DN was amplified.
[0287] The PCR products thus , obtained were digested with
restriction enzymes EcoRI and NotI, and then were inserted into an
EcoRI recognition site of an animal cell expression vector pCOS1
containing EF1-.alpha. promoter and the neomycin resistant gene to
produce an expression vector pTAK1DN. The expression vector pCOS1
was constructed by deleting the gene contained from plasmid
HEF-PMh-g.gamma.1 (see WO 92-19759) by digesting with EcoRI and Sam
I and then ligating the EcoRI-NotI-bamHI Adaptor (manufactured by
Takara Shuzo).
[0288] Then, pTAK1DN or pCOS1 that contains no inserted genes as a
control vector was linearized by digesting with a restriction
enzyme PvuI. These linearized vectors were introduced by
electroporation into human fibroblast-derived HT-1080 (ATCC CCL
121), a mouse kidney mesangial cell line SV40MES13 (ATCC CRL 1927),
and a mink pulmonary epithelial cell line Mv1Lu (ATCC CCL 64), and
cells into which the gene was introduced were selected using G418
(manufactured by GIBCO-BRL ).
[0289] Expression of each gene was confirmed by the RT-PCR method
using primer TA5 (SEQ ID NO: 20). and primer HG1-R1 (SEQ ID NO:
21). Thus, mRNA was isolated from the gene-introduced cells using
the Quick Prep mRNA Micro Purification kit (manufactured by
Pharmacia). Then using the First Strand cDNA Synthesis kit
(manufactured by Pharmacia), cDNA was synthesized from 150 ng of
mRNA. The introduction of the gene was confirmed using 5 .mu.l of
cDNA reaction mixture as a template.
Example 7
The Action of TGF-.beta. in Human Fibroblast-Derived HT-1080
[0290] The human fibroblast-derived HT-1080 cells (HT/DN2 and
HT/DN14) into which pTAK1DN had been introduced and the control
cells (HT/NEO) into which pCOS1 containing no inserted genes had
been introduced were incubated in a low-serum medium (Medium 199
containing 0.2% FBS; manufactured by GIBCO BRL) with or without 1
ng/ml TGF-.beta. (manufactured by King Jozo) for 24 hours. The
amount of fibronectin in the culture supernatant or the
extracellular matrix extract prepared using 1 M urea solution (1 M
urea, 1 mM DTT, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, Protease
inhibitor cocktail.(Complete.TM., manufactured by Boehringer
Mannheim)) was determined by the EIA method.
[0291] Thus, 100 .mu.l of the culture supernatant or the
extracellular matrix extract prepared using 1 M urea solution was
added to a 96-well microtiter plate (manufactured by Nunc), and the
plate was incubated overnight at 4.degree. C. After washing, it was
blocked using a 1% BSA solution (50 mM Tris-HCl, pH 8.0, 1 mM
MgCl.sub.2, 150 mM NaCl, 0.05% Tween 20, 0.02% sodium azide), and
then 10000 fold-diluted rabbit anti-human fibronectin antibody
(manufactured by CALBIOCHEM) was added to the above 1% BSA
solution, and was further incubated at room temperature for 2
hours.
[0292] After washing, alkaline phosphatase-labeled goat anti-rabbit
Ig antibody (manufactured by TAGO) was added, and was further
incubated at room temperature for one hour. Then the substrate
solution (p-nitrophenyl phosphate; manufactured by Sigma) was added
and absorbance at 450 nm was measured. As a standard, human
fibronectin (manufactured by Cappel) was used.
[0293] The results are shown in FIGS. 5A and B. In FIGS. 5A and B,
the numerical values indicate the mean +/- SD of the culture
supernatant and the extracellular matrix extracts prepared using 1
M urea, each prepared from 3 wells.
[0294] In the control cells HT/NEO, TGF-.beta. addition increased
fibronectin in the culture supernatant by about 6.1 fold, and that
in the extracellular matrix extract by about 11.4 fold. On the
other hand, in HT/DN2 and HT/DN14, cells that express TAK1-DN,
fibronectin in the culture supernatant increased by about 2.6 and
3.0 fold, respectively, and that in the extracellular matrix
extract increased by about 3.5 and 3.6 fold, respectively. These
results indicated that the production of fibronectin by TGF-.beta.
and the incorporation of fibronectin into the matrix were
suppressed by the expression of TAK1-DN.
Example 8
Effect of TGF-.beta. on Mouse Kidney Mesangial Cell Line
SV40MES13
[0295] The mouse mesangial cell line SV40MES13 (MES/DN3 and
MES/DN6) into which pTAK1DN had been introduced and the control
cells (MES/NEO) into which pCOS1 containing no inserted genes had
been introduced were incubated in a low-serum medium (Medium 199
containing 0.2% FBS) with or without 2.5 ng/ml TGF-.beta. for 24
hours. The amount of fibronectin in the culture supernatant or the
extracellular matrix extract prepared using 1 M urea solution was
determined by the EIA method as described in Example 7
[0296] The results are shown in FIGS. 6A and B. In FIGS. 6A and B.
the numerical values indicate the mean +/- SD of the culture
supernatant and the extracellular matrix extracts prepared using 1
M urea, each prepared from 3 wells. In the control cells MES/NEO,
TGF-.beta. addition increased fibronectin in the culture
supernatant by about 3.3 fold, and that in the extracellular matrix
extract by about 3.8 fold. On the other hand, in MED/DN3 and
MES/DN6, cells that express TAK1-DN, fibronectin in the culture
supernatant increased by about 2.4 and 2.3 fold, respectively, and
that in the extracellular matrix extract increased by about 2.6 and
2.1 fold, respectively. These results indicated that the production
of fibronectin by TGF-.beta. and the incorporation of fibronectin
into the matrix were suppressed by the expression of TAK1-DN.
[0297] Furthermore, the amount of type I and type IV collagen in
the culture supernatant was determined by the EIA method. Thus, 100
.mu.l of the culture supernatant was added to a 96-well microtiter
plate (manufactured by Nunc), and the plate was incubated overnight
at 4.degree. C. After washing, it was blocked using the above 1%
BSA solution, and then 5000-fold diluted rabbit anti-mouse type I
collagen antibody (manufactured by LSL) or rabbit anti-mouse type
IV collagen antibody (manufactured by LSL) was added, and was
further incubated at room temperature for 2 hours. After washing,
alkaline phosphatase-labeled goat anti-rabbit Ig antibody
(manufactured by TAGO) was added, and was further incubated at room
temperature for one hour. Then the substrate solution (manufactured
by Sigma) was added and absorbance at 450 nm was measured. As a
standard, mouse type I collagen (manufactured by Chemicon) or mouse
type IV collagen (manufactured by Cosmobio) was used.
[0298] The results are shown in FIGS. 7 and 8. In FIGS. 7 and 8,
the numerical values indicate the mean +/- SD of the culture
supernatant and the extracellular matrix extracts prepared using 1
M urea, each prepared from 3 wells. In the control cells MES/NEO,
TGF-.beta. addition increased type I collagen in the culture
supernatant by about 3.6 fold, and type IV collagen by about 2.0
fold. On the other hand, in MED/DN3 and MES4DN6, cells that express
TAK1-DN, type I collagen in the culture supernatant increased by
about 2.0 and 2.0 fold, respectively, and type IV collagen
increased by about 1.5 and 1.4 fold, respectively. These results
indicated that the production of type I collagen and type IV
collagen by TGF-.beta. was suppressed by the expression of
TAK1-DN.
Example 9
Effect of TGF-.beta. on Mink Epithelial Cell Line Mv1Lu
[0299] It is known that the cellular growth of mink epithelial cell
line Mv1Lu is stopped at the G1 phase by stimulation with
TGF-.beta. . Accordingly, effects on the inhibition of cellular
growth of TAK1-DN can be investigated using the mink epithelial
cell line Mv1Lu.
[0300] The mink epithelial cell line Mv1Lu (Mv/DN1 and Mv/DN4) into
which pTAK1DN had been introduced and the control cells (Mv/NEO)
into which pCOS1 containing no inserted genes had been introduced
were incubated in a low-serum medium (Medium 199 containing 0.2%
FBS) with or without various concentrations of TGF-.beta. for 24
hours. Then BrdU (manufactured by Boehringer Mannheim) was added to
a final concentration of 1 mM and was further incubated for 4
hours. Cellular growth could be evaluated by determining the amount
of BrdU incorporated into the cells according to the instructions
by the manufacturer.
Example 10
Construction of TAB1 Deletion Mutant Expression Vector
[0301] In order to determine the region in the TAB1 polypeptide
that is required for binding to the TAK1 polypeptide by the yeast
2-hybrid method, expression vectors for TAB1 deletion mutants were
constructed. Thus, it was designed that the TAB1 partial
polypeptide can be expressed as a fusion polypeptide with the GAL4
transcription activated domain polypeptide in yeast cells by
constructing a gene fragment encoding a partial polypeptide of the
TAB1 polypeptide, which is then inserted into a yeast 2-hybrid
expression vector pGAD10 (manufactured by CLONTECH), and was so
constructed.
Example 10-1
Construction of Deletion Mutants from the Amino Terminal
[0302] TAB1C45, TAB1C25, TAB1C24, TAB1C23, TAB1C22, TAB1C21 and
TAB1C20 are polypeptides that comprise 45, 25, 24, 23, 22, 21 and
20 amino acids, respectively, of the carboxy terminal of TAB1 (FIG.
11). An expression vector that expresses each of TAB1C45, TAB1C25,
TAB1C24, TAB1C23, TAB1C22, TAB1C21 and TAB1C20 as a fusion
polypeptide with the GAL4 transcription activated domain
polypeptide was constructed as follows:
[0303] The gene fragment that encodes TAB1C45 was amplified by the
PCR method using pGAD-TAB1 (Shibuya H. et al., Science (1996) 272,
1179-1182) as a template. pGAD-TAB1 is a yeast 2-hybrid expression
vector that expresses a fusion polypeptide of the 68 amino acids of
the carboxy terminal of TAB1 and the GAL4 transcription activated
domain polypeptide. Primers used are a sense primer TABC45 (SEQ ID
NO: 24) that contains a restriction enzyme XhoI recognition
sequence and an antisense primer TABCapEc (SEQ ID NO: 25) that
contains a restriction enzyme EcoRI recognition sequence and 2 stop
codons. The PCR method comprised a total of 15 cycles of 94.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
1 minute per cycle.
[0304] PCR reaction products were separated and purified by a 1%
low-melting point agarose gel (manufactured by Sigma), digested
with restriction enzymes XhoI and EcoRI, and then were inserted
into a yeast 2-hybrid expression vector pGAD-TAB1 (manufactured by
CLONTECH) to obtain a plasmid pGAD-TAB1C45 that expresses a fusion
polypeptide of TAB1C45 and the GAL4 transcription activated
domain.
[0305] A gene fragment that encodes TAB1C25 was amplified using a
sense primer C25X (SEQ ID NO: 26) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C25 that expresses a fusion polypeptide of
TAB1C25 and the GAL4 transcription activated domain was
obtained.
[0306] A gene fragment that encodes TAB1C24 was amplified using a
sense primer C24X (SEQ ID NO: 27) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C24 that expresses a fusion polypeptide of
TAB1C24 and the GAL4 transcription activated domain was
obtained.
[0307] A gene fragment that encodes TAB1C23 was amplified using a
sense primer C23X (SEQ ID NO: 28) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C23 that expresses a fusion polypeptide of
TAB1C23 and the GAL4 transcription activated domain was
obtained.
[0308] A gene fragment that encodes TAB1C22 was amplified using a
sense primer C22X (SEQ ID NO: 29) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C22 that expresses a fusion polypeptide of
TAB1C22 and the GAL4 transcription activated domain was
obtained.
[0309] A gene fragment that encodes TAB1C21 was amplified using a
sense primer C21X (SEQ ID NO: 30) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C21 that expresses a fusion polypeptide of
TAB1C21 and the GAL4 transcription activated domain was
obtained.
[0310] A gene fragment that encodes TAB1C20 was amplified using a
sense primer C20X (SEQ ID NO: 31) containing a restriction enzyme
XhoI recognition sequence and an antisense primer TABCapEc (SEQ ID
NO: 25) by the PCR method with plasmid pGAD-TAB1C45 as a template,
and plasmid pGAD-TAB1C20 that expresses a fusion polypeptide of
TAB1C20 and the GAL4 transcription activated domain was
obtained.
Example 10-2
Deletion from the Carboxy Terminal
[0311] Polypeptides that further lack polypeptides sequentially
from the carboxy terminal of the polypeptide comprising 45 amino
acids of the carboxy terminal portion of TAB1, i. e. TAB1C45
.DELTA.14, TAB1C45 .DELTA.19, TAB1C45 .DELTA.20, TAB1C45 .DELTA.21,
TAB1C45 .DELTA.22, TAB1C45 .DELTA.23, TAB1C45 .DELTA.24 and TAB1C45
.DELTA.25 (FIG. 12), were designed as follows:
[0312] Thus, TAB1C45 .DELTA.14 is a polypeptide that lacks 14 amino
acids from the carboxy terminal of the polypeptide comprising 45
amino acids of the carboxy terminal portion of TAB1. Similarly,
TAB1C45 .DELTA.19, TAB1C45 .DELTA.20, TAB1C45 .DELTA.21, TAB1C45
.DELTA.22, TAB1C45 .DELTA.23, TAB1C45 .DELTA.24 and TAB1C45
.DELTA.25 are polypeptides that lack 19, 20, 21, 22, 23, 24 and 25
amino acids, respectively, from the carboxy terminal of the
polypeptide comprising 45 amino acids of the carboxy terminal
portion of TAB1.
[0313] Plasmids that express these polypeptides as fusion
polypeptides with the GAL4 transcription activated domain were
constructed as follows:
[0314] The gene fragment that encodes TAB1C45 .DELTA.14 polypeptide
was amplified by the PCR method using pGAD-TAB1C45 as a template.
Thus, PCR was carried out using a sense primer TABC45 (SEQ ID NO:
24) that contains a restriction enzyme XhoI recognition sequence
and an antisense primer TABCD14A (SEQ ID NO: 32) that contains a
restriction enzyme EcoRI recognition sequence and 2 stop codons.
The PCR method comprised a total of 15 cycles of 94.degree. C. for
1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for 1
minute per cycle.
[0315] PCR reaction products were separated and purified by a 1%
low-melting point agarose gel (manufactured by Sigma), digested
with restriction enzymes XhoI and EcoRI, and then were inserted
into a yeast 2-hybrid expression vector pGAD10 (manufactured by
CLONTECH) to obtain a plasmid pGAD-TAB1C45D14 that expresses a
fusion polypeptide of TAB1C45 .DELTA.14 polypeptide and the GAL4
transcription activated domain.
[0316] A gene fragment that encodes TAB1C45 .DELTA.19 was amplified
using a sense primer TABC45 (SEQ ID NO: 24) and an antisense primer
TABCD19A (SEQ ID NO: 33) containing a restriction enzyme EcoRI
recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D19 that expresses a fusion polypeptide of TAB1C45
.DELTA.19 and the GAL4 transcription activated domain was
obtained.
[0317] A gene fragment that encodes TAB1C45 .DELTA.20 was amplified
using a sense primer TABC45 (SEQ ID NO: 24) and an antisense primer
TABCD20 (SEQ ID NO: 34) containing a restriction enzyme EcoRI
recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D20 that expresses a fusion polypeptide of TAB1C45
.DELTA.20 and the GAL4 transcription activated domain was
obtained.
[0318] A gene fragment that encodes TAB1C45 .DELTA.21 was amplified
using a sense primer TABC45 (SEQ ID NO: 24) and an antisense primer
TABCD21 (SEQ ID NO: 35) containing a restriction enzyme EcoRI
recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D21 that expresses a fusion polypeptide of TAB1C45
.DELTA.21 and the GAL4 transcription activated domain was
obtained.
[0319] A gene fragment that encodes TAB1C45 .DELTA.22 was amplified
using a sense primer TABC45 (SEQ ID NO: 24) and an antisense primer
TABCD22 (SEQ ID NO: 36) containing a restriction enzyme EcoRI
recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D22 that expresses a fusion polypeptide of TAB1C45
.DELTA.22 and the GAL4 transcription activated domain was
obtained.
[0320] A gene fragment that encodes TAB1C45 .DELTA.23 was amplified
using a sense primer. TABC45 (SEQ ID NO: 24) and an antisense
primer TABCD23 (SEQ ID NO: 37) containing a restriction enzyme
EcoRI recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D23 that expresses a fusion polypeptide of TAB1C45
.DELTA.23 and the GAL4 transcription activated domain was
obtained.
[0321] A gene fragment that encodes TAB1C45 .DELTA.24 was amplified
using a sense primer TABC45 (SEQ ID NO: 24) and an antisense primer
TABCD24 (SEQ ID NO: 38) containing a restriction enzyme EcoRI
recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D24 that expresses a fusion polypeptide of TAB1C45
.DELTA.24 and the GAL4 transcription activated domain was
obtained.
[0322] A gene fragment that encodes TAB1C45 .DELTA.25 was amplified
using a sense primer TABC45. (SEQ ID NO: 24) and an antisense
primer TABCD25 (SEQ ID NO: 39) containing a restriction enzyme
EcoRI recognition sequence and 2 stop codons by the PCR method with
plasmid pGAD-TAB1C45 as a template, and thereby plasmid
pGAD-TAB1C45D25 that expresses a fusion polypeptide of TAB1C45
.DELTA.25 and the , GAL4 transcription activated domain was
obtained.
Example 11
Transformation of Yeast
[0323] In order to evaluate each TAB1 deletion mutant constructed
in Example 10, a yeast 2-hybrid expression vector of each TAB1
deletion mutant and a yeast 2-hybrid expression vector pBTMHu11F
(Shibuya H. et al., Science (1996) 272, 1179-1182) that expresses
TAK1 were co-transformed into a yeast strain L40 (Shibuya H. et
al., Science (1996) 272, 1179-1182). One mg each of a TAB1 deletion
mutant expression vector (pGAD-TAB1C45 to pGAD-TAB1C45 in Working
Example 10-1 and pGAD-TAB1C45D14 to pGAD-TAB1C45D25 in Working
Example 10-2) or pGAD-TAB1 (Shibuya H. et al., Science (1996) 272,
1179-1182) as a control and pBTMHu11F were introduced into the L40
strain according to the instructions (MATCHMAKER.TM. Two-Hybrid
System, manufactured by CLONTECH), and were incubated on a
selection agar medium SD-ULW (glucose 20 g, agar (manufactured by
DIFCO) 20 g, Yeast Nitrogen Base w/o amino acids (manufactured by
DIFCO) 6.7 g, adenine 100 mg, isoleucine 30 mg, valine 150 mg,
arginine 20 mg, lysine 30 mg, methionine 20 mg, phenylalanine 50
mg, threonine 200 mg, tyrosine 30 mg, histidine 100 mg per liter of
medium) at 30.degree. C. for 3 days to obtain each
transformant.
Example 12
Evaluation of the Binding Ability of TAB1 Deletion Mutants to
TAK1
[0324] In order to evaluate the binding ability to TAK1 of each
TAB1 deletion mutant constructed in Example 10, activity was
determined by the yeast 2-hybrid method. Since a reporter gene lacZ
having the LexA binding sequence upstream thereof has been
integrated on the chromosome of the yeast strain L40, the binding
ability to TAK1 of each TAB1 deletion mutant can be evaluated in
relative terms by measuring the activity of .beta.-galactosidase
that is a reporter gene product.
Example 12-1
Evaluation of Deletion Mutants (TAB1C45 to TAB1C20) from the Amino
Terminal
[0325] The .beta.-galactosidase activity of each transformant
obtained in Example 11 was determined according to the instructions
(MATCHMAKER.TM. Two-Hybrid System, manufactured by CLONTECH), and
the activity was calculated using Miller Unit (Miller, J. H. (1972)
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.).
[0326] The results are shown in FIG. 11. In FIG. 11, the
.beta.-galactosidase activity of each TAB1 deletion mutant and the
yeast L40 transformed by the yeast 2-hybrid expression plasmid of
TAK1 was expressed in terms of Miller Units. Measurement was
carried out three times and the mean +/- SD is shown. The value
indicates a ratio based on the .beta.-galactosidase activity of the
yeast L40 transformed by TAB1C68 and the yeast 2-hybrid expression
plasmid of TAK1
[0327] Since specific activities of TAB1C25 and TAB1C24 are 0.28
and 0.35, respectively, whereas those of TAB1C23, TAB1C22, TAB1C21
and TAB1C20 markedly decrease to 0.05, 0.03, 0.03 and 0.03,
respectively, the amino terminal of the region required for binding
of TAB1 to TAK1 is believed to be the amino terminal Tyr residue
(amino acid position 481 in the amino acid sequence as set forth in
SEQ ID NO: 2) of TAB1C24.
Example 12-2
Evaluation of Deletion Mutants (TAB1C45 .DELTA.14 to TAB1C45
.DELTA.25) from the Carboxy Terminal
[0328] The .beta.-galactosidase activity of each transformant
obtained in Example 11 was determined according to the instructions
(MATCHMAKER.TM. Two-Hybrid System, manufactured by CLONTECH), and
the activity was calculated using Miller Units (Miller, J. H.
(1972) Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0329] The results are shown in FIG. 12. In FIG. 12, the
.beta.-galactosidase activity of the yeast L40 transformed by each
TAB1 deletion mutant and the yeast 2-hybrid expression plasmid of
TAK1 was expressed in terms of Miller Units. Measurement was
carried out three times and the mean +/- SD is shown. The value
indicates a ratio based on the .beta.-galactosidase activity of the
yeast L40 transformed by the TAB1C68 and yeast 2-hybrid expression
plasmid of TAK1.
[0330] Since specific activities of TAB1C45 .DELTA.19 and TAB1C45
.DELTA.20are 0.13 and 0.11, respectively, whereas those of TAB1C45
.DELTA.21, TAB1C45 .DELTA.22, TAB1C45 .DELTA.23, TAB1C45 .DELTA.24
and TAB1C45 .DELTA.25 markedly decrease to 0.01, 0.02, 0.00, 0.02
and 0.01, respectively, the carboxy terminal of the region required
for binding of TAB1 to TAK1 is believed to be the carboxy terminal
Phe residue (amino acid position 484 in the amino acid sequence as
set forth in SEQ ID NO: 2) of TAB1C45 .DELTA.20.
[0331] From the foregoing, the region required for binding of TAB1
to TAK1 is believed to be the region from Tyr at amino acid
position 481 to Phe at amino acid position 484 of the amino acid
sequence as set forth in SEQ ID NO: 2.
Example 13
Binding-Inhibition Study Using Synthetic Peptides
[0332] The fact that a synthetic peptide containing the amino acid
sequence identified as the TAK1 binding region can inhibit binding
between TAK1 and TAB1 was confirmed by the following experiment.
Thus, a peptide TAB1C-1 (SEQ ID NO: 40) comprising 16 amino acid
residues containing the TAK1 binding region of TAB1 described in
the above Example and a control peptide TAB1C-2 (SEQ ID NO: 41)
containing an amino acid sequence from Gln at position 437 to Gln
at position 451 of TAB1 were each synthesized, and were evaluated
for their effects on the binding between TAK1 and TAB1. The TAK1
and TAB1 used in the present invention were prepared in the
following manner. Thus, the TAK1 expression vector or the TAB1
expression vector, was introduced into COS-7 cells using
LIPOFECTOAMINE (manufactured by GIBCO-BRL) by a standard method.
After incubating for 72 hours, the cells were harvested and washed
with PBS. Subsequently, they were suspended in the lysis buffer (10
mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, Complete
Protease Inhibitor Cocktail (Boehringer Mannheim)), incubated at
4.degree. C. for 1 hour, and the insoluble components were removed
by centrifugation to prepare each cell extract.
[0333] The TAK1 expression vector pCOS-TAK1 was constructed as
described below. Thus, plasmid pBacTABF having a nucleotide
sequence encoding TAK1-6.times.His was digested with restriction
enzymes EcoRI and NotI, and a gene fragment containing a nucleotide
sequence encoding TAK1-6.times.His was purified using a 1.5%
low-melting point agarose gel (manufactured by Sigma) and was
inserted to an expression vector pCOS1 to construct pCOS-TAK1. The
TAB1 expression vector pCOS-FTAKB1 was constructed as described
below. Thus, a gene fragment encoding RLAG-TAB1 (SEQ ID NO: 42) to
which was added a FLAG tag comprising 8 amino acids to the amino
terminal of TAB1 was amplified by the PCR method using pBacTABF as
a template DNA. The PCR method was carried out as described above
using Eco-MetFTAB (SEQ ID NO: 44) as a sense primer that was
designed to contain a nucleotide sequence encoding a restriction
enzyme EcoRI recognition site, ATG initiation codon, and a FLAG tag
and TABC-Not (SEQ ID NO: 45) as an antisense primer that was
designed to contain a stop codon and a restriction enzyme NotI
recognition site. The PCR product was digested with restriction
enzymes EcoRI and NotI, inserted into pCOS1, and the plasmid that
has the correct base sequence was used as the expression vector
pCOS-FTAB1.
[0334] Subsequently, cell extracts each containing TAK1-6.times.His
or FLAG-TAB1 and a 5 mM synthetic peptide were mixed to a final
concentration of 50 or 500 mM, respectively, and then incubated
overnight at 4.degree. C. It was then immunoprecipitated using
anti-TAK1 antibody, and the amount of bound TAK1 and TAB1 was
evaluated by determining the amount of TAB1 that coprecipitated by
Western analysis. Thus, 2 mg each of anti-TAK1 polyclonal antibody
(manufactured by SantaCruz) was added to each reaction mixture, and
further incubated at 4.degree. C. for 1 hour. Then 40 .mu.l (50%
v/v) of Protein-G Sepharose (manufactured by Pharmacia) was added
thereto, and was further incubated for 1 hour. After the
immunoprecipitate was washed three times with TBS. containing 0.05%
Tween 20, it was subjected to SDS-PAGE. The immunoprecipitate
separated by SDS-PAGE was transferred to a nitrocellulose membrane
(manufactured by Schleicher & Schuell) and was subjected to a
Western analysis. After it was blocked using a TBS solution
containing 5% BSA, TAK1 in the immunoprecipitate was detected using
anti-TAK1 antibody (manufactured by SantaCruz) and the
coprecipitataed FLAG-TAB1 was detected using anti-FLAG M2 antibody
(manufactured by Kodak). The amount of TAK1 and FLAG-TAB1 was each
determined by quantifying each band using an analysis software
Quantity One (manufactured by PDI), and the amount of the
coprecipitated FLAG-TAB1 was corrected with the amount of TAK1 to
obtain the amount bound of TAK1 and TAB1.
[0335] The result is shown in FIG. 13. Compared to the , case
wherein no synthetic peptides were added, no decrease in binding
between TAK1 and TAB1 was observed when the control peptide TAB1C-2
was added, whereas a decrease in the amount of coprecipitated
FLAG-TAB1 was observed when TAB1C-1 was added. The above result
indicates that a synthetic peptide containing the TAK1 binding
region has an activity of inhibiting binding between TAK1 and TAB1.
Accordingly, it is believed that synthetic peptides containing the
amino acid sequence , identified as the TAK1 binding region or
derivatives thereof and substances that act on that region act on
binding between TAK1 and TAB1 and activate or inhibit signal
transduction from TAK1.
Example 14
Identification of the TAB1 Region Essential for the Induction of
TAK1 Activation
[0336] The region of TAB1 required to induce the kinase activity
after binding to TAK1 was identified using TAB1 deletion
mutants.
[0337] Expression vectors for the 40, 35 and 30 amino acid regions
(FIG. 14) in the carboxy terminal of TAB1 were constructed as
described below. Thus ,, genes encoding 40, 35 and 30 amino acid
regions respectively in the carboxy terminal of TAB1 were amplified
by the PCR method as described above, digested with restriction
enzymes XhoI and EcoRI, and inserted to the GAL4 transcription
activated domain expression vector pGAD10 to construct
pGAD-TAB1C40, pGAD-TAB1C35 and pGAD-TAB1C30, respectively.
Furthermore, expression vectors for the TAB1 deletion mutants (FIG.
14) comprising 68, 45 and 25 amino acid regions in the carboxy
terminal of TAB1 used were pGAD-TAB1, pGAD-TAB1C45 and
pGAD-TAB1C25.
[0338] The gene encoding the 40 amino acid region of the carboxy
terminal of TAB1 was amplified using a sense primer TABC40 (SEQ ID
NO: 46) containing a restriction enzyme XhoI recognition sequence
and an antisense primer TABCapEc (SEQ ID NO: 25) by the PCR method
with plasmid pGAD-TAB1C45 as a template DNA.
[0339] The gene encoding the 35 amino acid region of the carboxy
terminal of TAB1 was amplified using a sense primer TABC35 (SEQ ID
NO: 47) containing a restriction enzyme XhoI recognition sequence
and an antisense primer TABCapEc (SEQ ID NO: 25) by the PCR method
with plasmid pGAD-TAB1C45 as a template DNA.
[0340] The gene encoding the 30 amino acid region of the carboxy
terminal of TAB1 was amplified using a sense primer TABC30 (SEQ ID
NO: 48) containing a restriction enzyme XhoI recognition sequence
and an antisense primer TABCapEc (SEQ ID NO: 25) by the PCR method
with plasmid pGAD-TAB1C45 as a template DNA.
[0341] First, binding between the 68, 45, 40, 35, 30 and 25 amino
acid regions of the carboxy terminal of TAB1 and TAK1 was evaluated
by the yeast 2-hybrid method mentioned above.
[0342] Subsequently, it was investigated whether each TAB1 deletion
mutant can induce the kinase activity of TAK1 using the method
described in Japanese Unexamined Patent Publication (Kokai)
9(1997)-163990. Thus, the above TAB1 deletion mutant expression
vector and TAK1 expression vector pNVll-HUll (Yamaguchi, K. et al.,
Science (1995) 270, 2008-2011) were introduced into Saccharomyces
cereviceae SY1984-P strain (his3.DELTA., stell.DELTA., FUS1p:
:HIS3, STE7.sup.P368) to obtain deletion mutants. In this yeast
strain, the original his3 is lacking, and hence it can only grow
when foreign histidine is present in the medium or when the lacking
Stell activity is complemented by mutation. These transformants
were plated onto a SC-His (glucose 20 g, agar (manufactured by
DIFCO) 20 g, Yeast Nitrogen Base w/o amino acids (manufactured by
DIFCO) 6.7 g, adenine 100 mg, isoleucine 30 mg, valine 150 mg,
arginine 20 mg, lysine 30 mg, methionine 20 mg, phenylalanine 50
mg, threonine 200 mg, tyrosine 30 mg per liter of medium) plate
containing no histidine, incubated at 30.degree. C., and the growth
of yeast transformed with each expression vector was confirmed in
order to evaluate the ability of each TAB1 deletion mutant to
activate TAK1.
[0343] These results are shown in FIG. 14. Binding to TAK1 was
observed for all TAB1 deletion mutants whereas the ability to
activate TAK1 for each TAB1 mutant was observed for TAB1C68, 45,
40, 35 and 30 but not for TAB1C25. From these results, it is
believed that the region important for TAK1 activation is present
in between No. 30 and No. 26 (corresponding to Asp at position 475
to Glu at position 479 in the amino acid sequence of SEQ ID NO: 2)
from the carboxy terminal. Thus, peptides that lack an amino acid
sequence of this region, or peptides that contain this amino acid
sequence or derivatives thereof as well as substances that act on
this region can serve as an inhibitor or inducer or a stimulator of
TAK1 activation.
Reference Example 1
[0344] It was analyzed whether a polypeptide (TAK1-DN) comprising
Glu at position 77 to Gln at position 303 of the TAK1 polypeptide
as set forth in SEQ ID NO: 4 inhibits binding between the TAK1
polypeptide and the TAB1 polypeptide and whether it can inhibit,
the activation of the TAK1 polypeptide in animal cells by an animal
cell 2-hybrid system (Dang et al., (1991) Mol. Cell. Biol. 11,
954-962) using the TAB1 polypeptide and the TAK1 polypeptide.
[0345] First, a gene encoding a full-length TAK1 and TAK1-DN and a
gene encoding the GAL4 DNA-binding domain (GAL4-BD) were ligated to
construct an expression vector. A gene encoding a full-length TAK1
was prepared by digesting yeast 2-hybrid expression plasmid
pBTMHu11F (Shibuya H. et al., (1996) 272, 1179-1182) with
restriction enzymes EcoRI and PstI, and was then linked to the
EcoRI/Pst site of an expression vector pM (manufactured by
CLONTECH) containing the GAL4-BD gene, which was termed an animal
cell 2-hybrid expression plasmid pM-TAK1.
[0346] Subsequently, a gene encoding TAK1-DN was amplified using a
sense primer DNTAK5' (SEQ ID NO: 22) to which a restriction enzyme
EcoRI recognition site had been added and an antisense primer
DNTAK3' (SEQ ID NO: 23) to which a restriction enzyme PstI
recognition site had been added by PCR with plasmid pBTMHu11F as a
template DNA. After digestion with restriction enzymes EcoRI and
PstI, it was ligated to the pM vector to obtain an animal cell
2-hybrid expression plasmid pM-TAKlDN.
[0347] Then, a gene encoding TAB1C68 comprising 68 amino acid
residues of the carboxy terminal of the TAB1 polypeptide and a gene
encoding VP16 protein-derived transcription activated domain
(VP16-AD) of herpes simplex virus were ligated to construct an
expression vector. A gene encoding TAB1C68 was prepared by
digesting yeast 2-hybrid expression plasmid pGAD-TAB1 (Shibuya H.
et al., (1996) 272, 1179-1182) with a restriction enzyme EcoRI, and
was then linked to the EcoRI site of an expression vector pVP16
(manufactured by CLONTECH) containing a gene encoding VP16-AD,
which was termed an animal cell 2-hybrid expression plasmid
pVP16-C68.
[0348] The reporter plasmid used was pG-Luc in which a gene
encoding CAT of pG5CAT (manufactured by CLONTECH) having five
contiguous GAL4 binding sites and the chloramphenicol
asetyltransferase (CAT) gene downstream thereof replaced with the
luciferase gene.
[0349] After incubating overnight CHO cells (5.times.10.sup.4
cells/well), they were washed with PBS. Then a mixture comprising
500 ng of a GAL4-BD fusion protein expression plasmid (either of
pM, pM-TAK1, and pM-TAK1DN), 500 ng of a VP16-AD fusion protein
expression plasmid (either of pVp16 and pVp16-C68), 100 ng of the
reporter plasmid pG5-Luc and 50 ng of pRL-SV40 (containing the
luciferase gene of Renilla downstream of SV40 promoter:
manufactured by Promega) and 10 ml of LIPOFECTOAMINE (manufactured
by GIBCO-BRL) was added thereto and was incubated for 5 hours to
introduce genes.
[0350] After further incubating for 72 hours, luciferase activity
in each cell extract was determined using the Dual-LuciferaseTM
Assay System (manufactured by Promega). Thus, after the cells were
washed with PBS, 250 ml of the Passive Lysis Buffer was added
thereto, incubated at room temperature for 15 minutes , and 20
.mu.l of each was used as the cell extract for the assay. The
efficiency of gene introduction was corrected. with the measured
value of luciferase activity of Renilla by pRL-SV40. The result is
shown in FIG. 9.
[0351] Similarly to the combination of pM-TAK1 and pVP16-C68,
increases in luciferase activity was confirmed for the combination
of pM-TAK1DN and pVP16-C68, revealing that TAK1DN, as the
full-length TAK1, binds to TAB1.
Reference Example 2
[0352] Since TAK1DN does not contain lysine at position 63 of SEQ
ID NO: 2 that is an amino acid residue essential for ATP binding
for the TAK1 polypeptide to exhibit kinase activity, it is expected
to exhibit no kinase activity by itself. It is also expected to
inhibit the activation of endogenous TAK1 polypeptide through
inhibition of binding between the full-length TAK1 polypeptide and
the TAB1 polypeptide by binding to the TAB1 polypeptide in the
cell.
[0353] It has been demonstrated that PAI-1 (plasminogen activator
inhibitor type 1) expression is in creased due to stimulation by
TGF-.beta. in the Mv1Lu cell and that PAI-1 expression is inhibited
by forced expression of a catalytically inactive TAK1 polypeptide
mutant TAK1-K63W (Yamaguchi K. et al., (1995) Science 270,
2008-2011).
[0354] Accordingly, TAK1DN was subjected to forced expression in
the Mv1Lu cell to investigate the effects of TGF-.beta. stimulation
on PAI-1 expression. The TAK1DN expression vector used was the
above-mentioned TAK1DN, and the TAK1 polypeptide mutant TAK1-K63W
expression vector was constructed by inserting a gene (Yamaguchi K.
et al., (1995) Science 270, 2008-2011) encoding the TAK1
polypeptide mutant TAK1-K63W in which the lysine residue at
position 63 has been replaced with the tryptophan residue at the
EcoRI and NotI restriction enzyme site of pCOSl to give
pTAK1K63W.
[0355] The Mv1Lu cells into which pTAK1Dn had been introduced
(Mv/DN2), the cells into which pTAK1K63W had been introduced
(Mv/KN7), and the control cells into which pCOS1 containing no
inserted genes (Mv/NEO) had been introduced were each incubated in
a low-serum medium (MEM medium containing 0.2% FBS; manufactured by
GIBCO-BRL) with or without 10 ng/ml of TGF-.beta.1 for 24 hours.
The amount of PAI-1 in the culture supernatant was determined using
the PAI-1 Quantative ELISA (manufactured by CALBIOCHEM). The
results are shown in FIG. 10.
[0356] In the control cells there was an about 16-fold increase in
PAI-1 by TGF-.beta.1 addition in the culture supernatant, whereas
in the MV/KN6 cells that express the TAK1 polypeptide mutant
TAK1-K63W an increase in PAI-1 was about 6.5 fold and in the Mv/DN2
cells the increase was up to about 4.3 fold. Thus, TAK1DN inhibited
the effect of enhancing expression of PAI-1 by TGF-.beta.1
stimulation in a similar manner to the TAK1 polypeptide mutant
TAK1-K63W.
[0357] The forgoing has shown that TAK1DN inhibits signal
transduction via the TAK1 polypeptide and the TAB1 polypeptide by
TGF-.beta.1 stimulation, by inhibiting binding between the
endogenous TAK1 polypeptide and the TAB1 polypeptide.
[0358] Industrial Applicability
[0359] It was revealed that substances that inhibit binding between
the TAB1 polypeptide and the TAK1 polypeptide can be screened by
the screening method of the present invention. The screening method
of the present invention is useful for screening substances that
inhibit binding between the TAB1 polypeptide and the TAK1
polypeptide. Substances obtained by the screening method of the
present invention that inhibit binding between the TAB1 polypeptide
and the TAK1 polypeptide are useful as pharmaceutical agents.
[0360] Reference to the microorganisms deposited under the Patent
Cooperation Treaty, Rule 13-2, and the name of the Depository
Institute
[0361] Depository Institute
[0362] Name: the National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology Address:
1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan
[0363] Organism (1)
[0364] Indication: Escherichia coli MC1061/P3 (pEF-TAK1DN)
[0365] Accession number: FERM BP-5245
[0366] Date deposited: Sep. 28, 1995
[0367] Organism (2)
[0368] Indication: Escherichia coli MC1061/P3 (pEF-TAK1)
[0369] Accession number: FERM BP-5246
[0370] Date deposited: Sep. 28, 1995
[0371] Organism (3)
[0372] Indication: Escherichia coli HB101 (pBS-TAB1)
[0373] Accession number: FERM BP-5508
[0374] Date deposited: Apr. 19, 1996
[0375] Organism (4)
[0376] Indication: Escherichia coli JM109 (phTAK1)
[0377] Accession number: FERM BP-5598
[0378] Date deposited: Jul. 19, 1996
[0379] Organism (5)
[0380] Indication: Escherichia coli DH5a (TAB1-f-4)
[0381] Accession number: FERM BP-5599
[0382] Date deposited: Jul. 19, 1996
Sequence CWU 1
1
48 1 1560 DNA Homo sapiens CDS (30)..(1541) 1 gaattcgtgg cccgcagggt
tcctccaag atg gcg gcg cag agg agg agc ttg 53 Met Ala Ala Gln Arg
Arg Ser Leu 1 5 ctg cag agt gag cag cag cca agc tgg aca gat gac ctg
cct ctc tgc 101 Leu Gln Ser Glu Gln Gln Pro Ser Trp Thr Asp Asp Leu
Pro Leu Cys 10 15 20 cac ctc tct ggg gtt ggc tca gcc tcc aac cgc
agc tac tct gct gat 149 His Leu Ser Gly Val Gly Ser Ala Ser Asn Arg
Ser Tyr Ser Ala Asp 25 30 35 40 ggc aag ggc act gag agc cac ccg cca
gag gac agc tgg ctc aag ttc 197 Gly Lys Gly Thr Glu Ser His Pro Pro
Glu Asp Ser Trp Leu Lys Phe 45 50 55 agg agt gag aac aac tgc ttc
ctg tat ggg gtc ttc aac ggc tat gat 245 Arg Ser Glu Asn Asn Cys Phe
Leu Tyr Gly Val Phe Asn Gly Tyr Asp 60 65 70 ggc aac cga gtg acc
aac ttc gtg gcc cag cgg ctg tcc gca gag ctc 293 Gly Asn Arg Val Thr
Asn Phe Val Ala Gln Arg Leu Ser Ala Glu Leu 75 80 85 ctg ctg ggc
cag ctg aat gcc gag cac gcc gag gcc gat gtg cgg cgt 341 Leu Leu Gly
Gln Leu Asn Ala Glu His Ala Glu Ala Asp Val Arg Arg 90 95 100 gtg
ctg ctg cag gcc ttc gat gtg gtg gag agg agc ttc ctg gag tcc 389 Val
Leu Leu Gln Ala Phe Asp Val Val Glu Arg Ser Phe Leu Glu Ser 105 110
115 120 att gac gac gcc ttg gct gag aag gca agc ctc cag tcg caa ttg
cca 437 Ile Asp Asp Ala Leu Ala Glu Lys Ala Ser Leu Gln Ser Gln Leu
Pro 125 130 135 gag gga gtc cct cag cac cag ctg cct cct cag tat cag
aag atc ctt 485 Glu Gly Val Pro Gln His Gln Leu Pro Pro Gln Tyr Gln
Lys Ile Leu 140 145 150 gag aga ctc aag acg tta gag agg gaa att tcg
gga ggg gcc atg gcc 533 Glu Arg Leu Lys Thr Leu Glu Arg Glu Ile Ser
Gly Gly Ala Met Ala 155 160 165 gtt gtg gcg gtc ctt ctc aac aac aag
ctc tac gtc gcc aat gtc ggt 581 Val Val Ala Val Leu Leu Asn Asn Lys
Leu Tyr Val Ala Asn Val Gly 170 175 180 aca aac cgt gca ctt tta tgc
aaa tcg aca gtg gat ggg ttg cag gtg 629 Thr Asn Arg Ala Leu Leu Cys
Lys Ser Thr Val Asp Gly Leu Gln Val 185 190 195 200 aca cag ctg aac
gtg gac cac acc aca gag aac gag gat gag ctc ttc 677 Thr Gln Leu Asn
Val Asp His Thr Thr Glu Asn Glu Asp Glu Leu Phe 205 210 215 cgt ctt
tcg cag ctg ggc ttg gat gct gga aag atc aag cag gtg ggg 725 Arg Leu
Ser Gln Leu Gly Leu Asp Ala Gly Lys Ile Lys Gln Val Gly 220 225 230
atc atc tgt ggg cag gag agc acc cgg cgg atc ggg gat tac aag gtt 773
Ile Ile Cys Gly Gln Glu Ser Thr Arg Arg Ile Gly Asp Tyr Lys Val 235
240 245 aaa tat ggc tac acg gac att gac ctt ctc agc gct gcc aag tcc
aaa 821 Lys Tyr Gly Tyr Thr Asp Ile Asp Leu Leu Ser Ala Ala Lys Ser
Lys 250 255 260 cca atc atc gca gag cca gaa atc cat ggg gca cag ccg
ctg gat ggg 869 Pro Ile Ile Ala Glu Pro Glu Ile His Gly Ala Gln Pro
Leu Asp Gly 265 270 275 280 gtg acg ggc ttc ttg gtg ctg atg tcg gag
ggg ttg tac aag gcc cta 917 Val Thr Gly Phe Leu Val Leu Met Ser Glu
Gly Leu Tyr Lys Ala Leu 285 290 295 gag gca gcc cat ggg cct ggg cag
gcc aac cag gag att gct gcg atg 965 Glu Ala Ala His Gly Pro Gly Gln
Ala Asn Gln Glu Ile Ala Ala Met 300 305 310 att gac act gag ttt gcc
aag cag acc tcc ctg gac gca gtg gcc cag 1013 Ile Asp Thr Glu Phe
Ala Lys Gln Thr Ser Leu Asp Ala Val Ala Gln 315 320 325 gcc gtc gtg
gac cgg gtg aag cgc atc cac agc gac acc ttc gcc agt 1061 Ala Val
Val Asp Arg Val Lys Arg Ile His Ser Asp Thr Phe Ala Ser 330 335 340
ggt ggg gag cgt gcc agg ttc tgc ccc cgg cac gag gac atg acc ctg
1109 Gly Gly Glu Arg Ala Arg Phe Cys Pro Arg His Glu Asp Met Thr
Leu 345 350 355 360 cta gtg agg aac ttt ggc tac ccg ctg ggc gaa atg
agc cag ccc aca 1157 Leu Val Arg Asn Phe Gly Tyr Pro Leu Gly Glu
Met Ser Gln Pro Thr 365 370 375 ccg agc cca gcc cca gct gca gga gga
cga gtg tac cct gtg tct gtg 1205 Pro Ser Pro Ala Pro Ala Ala Gly
Gly Arg Val Tyr Pro Val Ser Val 380 385 390 cca tac tcc agc gcc cag
agc acc agc aag acc agc gtg acc ctc tcc 1253 Pro Tyr Ser Ser Ala
Gln Ser Thr Ser Lys Thr Ser Val Thr Leu Ser 395 400 405 ctt gtc atg
ccc tcc cag ggc cag atg gtc aac ggg gct cac agt gct 1301 Leu Val
Met Pro Ser Gln Gly Gln Met Val Asn Gly Ala His Ser Ala 410 415 420
tcc acc ctg gac gaa gcc acc ccc acc ctc acc aac caa agc ccg acc
1349 Ser Thr Leu Asp Glu Ala Thr Pro Thr Leu Thr Asn Gln Ser Pro
Thr 425 430 435 440 tta acc ctg cag tcc acc aac acg cac acg cag agc
agc agc tcc agc 1397 Leu Thr Leu Gln Ser Thr Asn Thr His Thr Gln
Ser Ser Ser Ser Ser 445 450 455 tct gac gga ggc ctc ttc cgc tcc cgg
ccc gcc cac tcg ctc ccg cct 1445 Ser Asp Gly Gly Leu Phe Arg Ser
Arg Pro Ala His Ser Leu Pro Pro 460 465 470 ggc gag gac ggt cgt gtt
gag ccc tat gtg gac ttt gct gag ttt tac 1493 Gly Glu Asp Gly Arg
Val Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr 475 480 485 cgc ctc tgg
agc gtg gac cat ggc gag cag agc gtg gtg aca gca ccg 1541 Arg Leu
Trp Ser Val Asp His Gly Glu Gln Ser Val Val Thr Ala Pro 490 495 500
tagggcagcc ggaggaatg 1560 2 504 PRT Homo sapiens 2 Met Ala Ala Gln
Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser 1 5 10 15 Trp Thr
Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala 20 25 30
Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His Pro 35
40 45 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys Phe
Leu 50 55 60 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr
Asn Phe Val 65 70 75 80 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly
Gln Leu Asn Ala Glu 85 90 95 His Ala Glu Ala Asp Val Arg Arg Val
Leu Leu Gln Ala Phe Asp Val 100 105 110 Val Glu Arg Ser Phe Leu Glu
Ser Ile Asp Asp Ala Leu Ala Glu Lys 115 120 125 Ala Ser Leu Gln Ser
Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu 130 135 140 Pro Pro Gln
Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg 145 150 155 160
Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu Asn Asn 165
170 175 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu Cys
Lys 180 185 190 Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn Val
Asp His Thr 195 200 205 Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser
Gln Leu Gly Leu Asp 210 215 220 Ala Gly Lys Ile Lys Gln Val Gly Ile
Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg Arg Ile Gly Asp Tyr
Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 Leu Leu Ser Ala
Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270 His Gly
Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275 280 285
Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln 290
295 300 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys
Gln 305 310 315 320 Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp
Arg Val Lys Arg 325 330 335 Ile His Ser Asp Thr Phe Ala Ser Gly Gly
Glu Arg Ala Arg Phe Cys 340 345 350 Pro Arg His Glu Asp Met Thr Leu
Leu Val Arg Asn Phe Gly Tyr Pro 355 360 365 Leu Gly Glu Met Ser Gln
Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly 370 375 380 Gly Arg Val Tyr
Pro Val Ser Val Pro Tyr Ser Ser Ala Gln Ser Thr 385 390 395 400 Ser
Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln Gly Gln 405 410
415 Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu Ala Thr Pro
420 425 430 Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr
Asn Thr 435 440 445 His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly
Leu Phe Arg Ser 450 455 460 Arg Pro Ala His Ser Leu Pro Pro Gly Glu
Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr Val Asp Phe Ala Glu Phe
Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 Glu Gln Ser Val Val
Thr Ala Pro 500 3 2656 DNA Homo sapiens CDS (183)..(1919) 3
gtcgagatcc attgtgctct aaagacggct gtggccgctg cctctacccc cgccacggat
60 cgccgggtag taggactgcg cggctccagg ctgagggtcg gtccggaggc
gggtgggcgc 120 gggtctcacc cggattgtcc gggtggcacc gttcccggcc
ccaccgggcg ccgcgaggga 180 tc atg tct aca gcc tct gcc gcc tcc tcc
tcc tcc tcg tct tcg gcc 227 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser
Ser Ser Ser Ser Ala 1 5 10 15 ggt gag atg atc gaa gcc cct tcc cag
gtc ctc aac ttt gaa gag atc 275 Gly Glu Met Ile Glu Ala Pro Ser Gln
Val Leu Asn Phe Glu Glu Ile 20 25 30 gac tac aag gag atc gag gtg
gaa gag gtt gtt gga aga gga gcc ttt 323 Asp Tyr Lys Glu Ile Glu Val
Glu Glu Val Val Gly Arg Gly Ala Phe 35 40 45 gga gtt gtt tgc aaa
gct aag tgg aga gca aaa gat gtt gct att aaa 371 Gly Val Val Cys Lys
Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys 50 55 60 caa ata gaa
agt gaa tct gag agg aaa gcg ttt att gta gag ctt cgg 419 Gln Ile Glu
Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg 65 70 75 cag
tta tcc cgt gtg aac cat cct aat att gta aag ctt tat gga gcc 467 Gln
Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala 80 85
90 95 tgc ttg aat cca gtg tgt ctt gtg atg gaa tat gct gaa ggg ggc
tct 515 Cys Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly
Ser 100 105 110 tta tat aat gtg ctg cat ggt gct gaa cca ttg cca tat
tat act gct 563 Leu Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr
Tyr Thr Ala 115 120 125 gcc cac gca atg agt tgg tgt tta cag tgt tcc
caa gga gtg gct tat 611 Ala His Ala Met Ser Trp Cys Leu Gln Cys Ser
Gln Gly Val Ala Tyr 130 135 140 ctt cac agc atg caa ccc aaa gcg cta
att cac agg gac ctg aaa cca 659 Leu His Ser Met Gln Pro Lys Ala Leu
Ile His Arg Asp Leu Lys Pro 145 150 155 cca aac tta ctg ctg gtt gca
ggg ggg aca gtt cta aaa att tgt gat 707 Pro Asn Leu Leu Leu Val Ala
Gly Gly Thr Val Leu Lys Ile Cys Asp 160 165 170 175 ttt ggt aca gcc
tgt gac att cag aca cac atg acc aat aac aag ggg 755 Phe Gly Thr Ala
Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly 180 185 190 agt gct
gct tgg atg gca cct gaa gtt ttt gaa ggt agt aat tac agt 803 Ser Ala
Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser 195 200 205
gaa aaa tgt gac gtc ttc agc tgg ggt att att ctt tgg gaa gtg ata 851
Glu Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile 210
215 220 acg cgt cgg aaa ccc ttt gat gag att ggt ggc cca gct ttc cga
atc 899 Thr Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg
Ile 225 230 235 atg tgg gct gtt cat aat ggt act cga cca cca ctg ata
aaa aat tta 947 Met Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile
Lys Asn Leu 240 245 250 255 cct aag ccc att gag agc ctg atg act cgt
tgt tgg tct aaa gat cct 995 Pro Lys Pro Ile Glu Ser Leu Met Thr Arg
Cys Trp Ser Lys Asp Pro 260 265 270 tcc cag cgc cct tca atg gag gaa
att gtg aaa ata atg act cac ttg 1043 Ser Gln Arg Pro Ser Met Glu
Glu Ile Val Lys Ile Met Thr His Leu 275 280 285 atg cgg tac ttt cca
gga gca gat gag cca tta cag tat cct tgt cag 1091 Met Arg Tyr Phe
Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln 290 295 300 tat tca
gat gaa gga cag agc aac tct gcc acc agt aca ggc tca ttc 1139 Tyr
Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser Phe 305 310
315 atg gac att gct tct aca aat acg agt aac aaa agt gac act aat atg
1187 Met Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn
Met 320 325 330 335 gag caa gtt cct gcc aca aat gat act att aag cgc
tta gaa tca aaa 1235 Glu Gln Val Pro Ala Thr Asn Asp Thr Ile Lys
Arg Leu Glu Ser Lys 340 345 350 ttg ttg aaa aat cag gca aag caa cag
agt gaa tct gga cgt tta agc 1283 Leu Leu Lys Asn Gln Ala Lys Gln
Gln Ser Glu Ser Gly Arg Leu Ser 355 360 365 ttg gga gcc tcc cat ggg
agc agt gtg gag agc ttg ccc cca acc tct 1331 Leu Gly Ala Ser His
Gly Ser Ser Val Glu Ser Leu Pro Pro Thr Ser 370 375 380 gag ggc aag
agg atg agt gct gac atg tct gaa ata gaa gct agg atc 1379 Glu Gly
Lys Arg Met Ser Ala Asp Met Ser Glu Ile Glu Ala Arg Ile 385 390 395
gcc gca acc aca ggc aac gga cag cca aga cgt aga tcc atc caa gac
1427 Ala Ala Thr Thr Gly Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln
Asp 400 405 410 415 ttg act gta act gga aca gaa cct ggt cag gtg agc
agt agg tca tcc 1475 Leu Thr Val Thr Gly Thr Glu Pro Gly Gln Val
Ser Ser Arg Ser Ser 420 425 430 agt ccc agt gtc aga atg att act acc
tca gga cca acc tca gaa aag 1523 Ser Pro Ser Val Arg Met Ile Thr
Thr Ser Gly Pro Thr Ser Glu Lys 435 440 445 cca act cga agt cat cca
tgg acc cct gat gat tcc aca gat acc aat 1571 Pro Thr Arg Ser His
Pro Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn 450 455 460 gga tca gat
aac tcc atc cca atg gct tat ctt aca ctg gat cac caa 1619 Gly Ser
Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr Leu Asp His Gln 465 470 475
cta cag cct cta gca ccg tgc cca aac tcc aaa gaa tct atg gca gtg
1667 Leu Gln Pro Leu Ala Pro Cys Pro Asn Ser Lys Glu Ser Met Ala
Val 480 485 490 495 ttt gaa cag cat tgt aaa atg gca caa gaa tat atg
aaa gtt caa aca 1715 Phe Glu Gln His Cys Lys Met Ala Gln Glu Tyr
Met Lys Val Gln Thr 500 505 510 gaa att gca ttg tta tta cag aga aag
caa gaa cta gtt gca gaa ctg 1763 Glu Ile Ala Leu Leu Leu Gln Arg
Lys Gln Glu Leu Val Ala Glu Leu 515 520 525 gac cag gat gaa aag gac
cag caa aat aca tct cgc ctg gta cag gaa 1811 Asp Gln Asp Glu Lys
Asp Gln Gln Asn Thr Ser Arg Leu Val Gln Glu 530 535 540 cat aaa aag
ctt tta gat gaa aac aaa agc ctt tct act tac tac cag 1859 His Lys
Lys Leu Leu Asp Glu Asn Lys Ser Leu Ser Thr Tyr Tyr Gln 545 550 555
caa tgc aaa aaa caa cta gag gtc atc aga agt cag cag cag aaa cga
1907 Gln Cys Lys Lys Gln Leu Glu Val Ile Arg Ser Gln Gln Gln Lys
Arg 560 565 570 575 caa ggc act tca tgattctctg ggaccgttac
attttgaaat atgcaaagaa 1959 Gln Gly Thr Ser agactttttt tttaaggaaa
ggaaaacctt ataatgacga ttcatgagtg ttagcttttt 2019 ggcgtgttct
gaatgccaac tgcctatatt tgctgcattt ttttcattgt ttattttcct 2079
tttctcatgg tggacataca attttactgt ttcattgcat aacatggtag catctgtgac
2139 ttgaatgagc agcactttgc aacttcaaaa cagatgcagt gaactgtggc
tgtatatgca 2199 tgctcattgt gtgaaggcta gcctaacaga acaggaggta
tcaaactagc tgctatgtgc 2259 aaacagcgtc cattttttca tattagaggt
ggaacctcaa gaatgacttt attcttgtat 2319 ctcatctcaa aatattaata
atttttttcc caaaagatgg tatataccaa gttaaagaca 2379 gggtattata
aatttagagt gattggtggt atattacgga aatacggaac ctttagggat 2439
agttccgtgt aagggctttg atgccagcat ccttggatca gtactgaact cagttccatc
2499 cgtaaaatat gtaaaggtaa gtggcagctg ctctatttaa tgaaagcagt
tttaccggat 2559 tttgttagac taaaatttga ttgtgataca ttgaacaaaa
tggaactcat tttttttaag 2619 gagtaaagat tttctttaga gcacaatgga tctcgac
2656 4 579 PRT Homo sapiens 4 Met Ser Thr Ala Ser Ala Ala Ser Ser
Ser
Ser Ser Ser Ser Ala Gly 1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln
Val Leu Asn Phe Glu Glu Ile Asp 20 25 30 Tyr Lys Glu Ile Glu Val
Glu Glu Val Val Gly Arg Gly Ala Phe Gly 35 40 45 Val Val Cys Lys
Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln 50 55 60 Ile Glu
Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln 65 70 75 80
Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys 85
90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser
Leu 100 105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr
Thr Ala Ala 115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln
Gly Val Ala Tyr Leu 130 135 140 His Ser Met Gln Pro Lys Ala Leu Ile
His Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala
Gly Gly Thr Val Leu Lys Ile Cys Asp Phe 165 170 175 Gly Thr Ala Cys
Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser 180 185 190 Ala Ala
Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu 195 200 205
Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr 210
215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile
Met 225 230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile
Lys Asn Leu Pro 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys
Trp Ser Lys Asp Pro Ser 260 265 270 Gln Arg Pro Ser Met Glu Glu Ile
Val Lys Ile Met Thr His Leu Met 275 280 285 Arg Tyr Phe Pro Gly Ala
Asp Glu Pro Leu Gln Tyr Pro Cys Gln Tyr 290 295 300 Ser Asp Glu Gly
Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser Phe Met 305 310 315 320 Asp
Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn Met Glu 325 330
335 Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg Leu Glu Ser Lys Leu
340 345 350 Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu Ser Gly Arg Leu
Ser Leu 355 360 365 Gly Ala Ser His Gly Ser Ser Val Glu Ser Leu Pro
Pro Thr Ser Glu 370 375 380 Gly Lys Arg Met Ser Ala Asp Met Ser Glu
Ile Glu Ala Arg Ile Ala 385 390 395 400 Ala Thr Thr Gly Asn Gly Gln
Pro Arg Arg Arg Ser Ile Gln Asp Leu 405 410 415 Thr Val Thr Gly Thr
Glu Pro Gly Gln Val Ser Ser Arg Ser Ser Ser 420 425 430 Pro Ser Val
Arg Met Ile Thr Thr Ser Gly Pro Thr Ser Glu Lys Pro 435 440 445 Thr
Arg Ser His Pro Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn Gly 450 455
460 Ser Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr Leu Asp His Gln Leu
465 470 475 480 Gln Pro Leu Ala Pro Cys Pro Asn Ser Lys Glu Ser Met
Ala Val Phe 485 490 495 Glu Gln His Cys Lys Met Ala Gln Glu Tyr Met
Lys Val Gln Thr Glu 500 505 510 Ile Ala Leu Leu Leu Gln Arg Lys Gln
Glu Leu Val Ala Glu Leu Asp 515 520 525 Gln Asp Glu Lys Asp Gln Gln
Asn Thr Ser Arg Leu Val Gln Glu His 530 535 540 Lys Lys Leu Leu Asp
Glu Asn Lys Ser Leu Ser Thr Tyr Tyr Gln Gln 545 550 555 560 Cys Lys
Lys Gln Leu Glu Val Ile Arg Ser Gln Gln Gln Lys Arg Gln 565 570 575
Gly Thr Ser 5 8 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 5 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 6
6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 6 His His His His His His 1 5 7 27 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 7
ccggaattca tggcggcgca gaggagg 27 8 72 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 8 agctctagat
cattatttat cgtcatcgtc tttgtagtca gaacctccgg tacccggtgc 60
tgtcaccacg ct 72 9 5 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 9 Gly Thr Gly Gly Ser 1 5 10
1569 DNA Homo sapiens CDS (7)..(1557) 10 gaattc atg gcg gcg cag agg
agg agc ttg ctg cag agt gag cag cag 48 Met Ala Ala Gln Arg Arg Ser
Leu Leu Gln Ser Glu Gln Gln 1 5 10 cca agc tgg aca gat gac ctg cct
ctc tgc cac ctc tct ggg gtt ggc 96 Pro Ser Trp Thr Asp Asp Leu Pro
Leu Cys His Leu Ser Gly Val Gly 15 20 25 30 tca gcc tcc aac cgc agc
tac tct gct gat ggc aag ggc act gag agc 144 Ser Ala Ser Asn Arg Ser
Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser 35 40 45 cac ccg cca gag
gac agc tgg ctc aag ttc agg agt gag aac aac tgc 192 His Pro Pro Glu
Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys 50 55 60 ttc ctg
tat ggg gtc ttc aac ggc tat gat ggc aac cga gtg acc aac 240 Phe Leu
Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr Asn 65 70 75
ttc gtg gcc cag cgg ctg tcc gca gag ctc ctg ctg ggc cag ctg aat 288
Phe Val Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn 80
85 90 gcc gag cac gcc gag gcc gat gtg cgg cgt gtg ctg ctg cag gcc
ttc 336 Ala Glu His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala
Phe 95 100 105 110 gat gtg gtg gag agg agc ttc ctg gag tcc att gac
gac gcc ttg gct 384 Asp Val Val Glu Arg Ser Phe Leu Glu Ser Ile Asp
Asp Ala Leu Ala 115 120 125 gag aag gca agc ctc cag tcg caa ttg cca
gag gga gtc cct cag cac 432 Glu Lys Ala Ser Leu Gln Ser Gln Leu Pro
Glu Gly Val Pro Gln His 130 135 140 cag ctg cct cct cag tat cag aag
atc ctt gag aga ctc aag acg tta 480 Gln Leu Pro Pro Gln Tyr Gln Lys
Ile Leu Glu Arg Leu Lys Thr Leu 145 150 155 gag agg gaa att tcg gga
ggg gcc atg gcc gtt gtg gcg gtc ctt ctc 528 Glu Arg Glu Ile Ser Gly
Gly Ala Met Ala Val Val Ala Val Leu Leu 160 165 170 aac aac aag ctc
tac gtc gcc aat gtc ggt aca aac cgt gca ctt tta 576 Asn Asn Lys Leu
Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu 175 180 185 190 tgc
aaa tcg aca gtg gat ggg ttg cag gtg aca cag ctg aac gtg gac 624 Cys
Lys Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn Val Asp 195 200
205 cac acc aca gag aac gag gat gag ctc ttc cgt ctt tcg cag ctg ggc
672 His Thr Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser Gln Leu Gly
210 215 220 ttg gat gct gga aag atc aag cag gtg ggg atc atc tgt ggg
cag gag 720 Leu Asp Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys Gly
Gln Glu 225 230 235 agc acc cgg cgg atc ggg gat tac aag gtt aaa tat
ggc tac acg gac 768 Ser Thr Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr
Gly Tyr Thr Asp 240 245 250 att gac ctt ctc agc gct gcc aag tcc aaa
cca atc atc gca gag cca 816 Ile Asp Leu Leu Ser Ala Ala Lys Ser Lys
Pro Ile Ile Ala Glu Pro 255 260 265 270 gaa atc cat ggg gca cag ccg
ctg gat ggg gtg acg ggc ttc ttg gtg 864 Glu Ile His Gly Ala Gln Pro
Leu Asp Gly Val Thr Gly Phe Leu Val 275 280 285 ctg atg tcg gag ggg
ttg tac aag gcc cta gag gca gcc cat ggg cct 912 Leu Met Ser Glu Gly
Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro 290 295 300 ggg cag gcc
aac cag gag att gct gcg atg att gac act gag ttt gcc 960 Gly Gln Ala
Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala 305 310 315 aag
cag acc tcc ctg gac gca gtg gcc cag gcc gtc gtg gac cgg gtg 1008
Lys Gln Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg Val 320
325 330 aag cgc atc cac agc gac acc ttc gcc agt ggt ggg gag cgt gcc
agg 1056 Lys Arg Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg
Ala Arg 335 340 345 350 ttc tgc ccc cgg cac gag gac atg acc ctg cta
gtg agg aac ttt ggc 1104 Phe Cys Pro Arg His Glu Asp Met Thr Leu
Leu Val Arg Asn Phe Gly 355 360 365 tac ccg ctg ggc gaa atg agc cag
ccc aca ccg agc cca gcc cca gct 1152 Tyr Pro Leu Gly Glu Met Ser
Gln Pro Thr Pro Ser Pro Ala Pro Ala 370 375 380 gca gga gga cga gtg
tac cct gtg tct gtg cca tac tcc agc gcc cag 1200 Ala Gly Gly Arg
Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala Gln 385 390 395 agc acc
agc aag acc agc gtg acc ctc tcc ctt gtc atg ccc tcc cag 1248 Ser
Thr Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln 400 405
410 ggc cag atg gtc aac ggg gct cac agt gct tcc acc ctg gac gaa gcc
1296 Gly Gln Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu
Ala 415 420 425 430 acc ccc acc ctc acc aac caa agc ccg acc tta acc
ctg cag tcc acc 1344 Thr Pro Thr Leu Thr Asn Gln Ser Pro Thr Leu
Thr Leu Gln Ser Thr 435 440 445 aac acg cac acg cag agc agc agc tcc
agc tct gac gga ggc ctc ttc 1392 Asn Thr His Thr Gln Ser Ser Ser
Ser Ser Ser Asp Gly Gly Leu Phe 450 455 460 cgc tcc cgg ccc gcc cac
tcg ctc ccg cct ggc gag gac ggt cgt gtt 1440 Arg Ser Arg Pro Ala
His Ser Leu Pro Pro Gly Glu Asp Gly Arg Val 465 470 475 gag ccc tat
gtg gac ttt gct gag ttt tac cgc ctc tgg agc gtg gac 1488 Glu Pro
Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp 480 485 490
cat ggc gag cag agc gtg gtg aca gca ccg ggt acc gga ggt tct gac
1536 His Gly Glu Gln Ser Val Val Thr Ala Pro Gly Thr Gly Gly Ser
Asp 495 500 505 510 tac aaa gac gat gac gat aaa taatgatcta ga 1569
Tyr Lys Asp Asp Asp Asp Lys 515 11 517 PRT Homo sapiens 11 Met Ala
Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser 1 5 10 15
Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala 20
25 30 Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His
Pro 35 40 45 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn
Cys Phe Leu 50 55 60 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg
Val Thr Asn Phe Val 65 70 75 80 Ala Gln Arg Leu Ser Ala Glu Leu Leu
Leu Gly Gln Leu Asn Ala Glu 85 90 95 His Ala Glu Ala Asp Val Arg
Arg Val Leu Leu Gln Ala Phe Asp Val 100 105 110 Val Glu Arg Ser Phe
Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys 115 120 125 Ala Ser Leu
Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu 130 135 140 Pro
Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg 145 150
155 160 Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu Asn
Asn 165 170 175 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu
Leu Cys Lys 180 185 190 Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu
Asn Val Asp His Thr 195 200 205 Thr Glu Asn Glu Asp Glu Leu Phe Arg
Leu Ser Gln Leu Gly Leu Asp 210 215 220 Ala Gly Lys Ile Lys Gln Val
Gly Ile Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg Arg Ile Gly
Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 Leu Leu
Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270
His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275
280 285 Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly
Gln 290 295 300 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe
Ala Lys Gln 305 310 315 320 Thr Ser Leu Asp Ala Val Ala Gln Ala Val
Val Asp Arg Val Lys Arg 325 330 335 Ile His Ser Asp Thr Phe Ala Ser
Gly Gly Glu Arg Ala Arg Phe Cys 340 345 350 Pro Arg His Glu Asp Met
Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro 355 360 365 Leu Gly Glu Met
Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly 370 375 380 Gly Arg
Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala Gln Ser Thr 385 390 395
400 Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln Gly Gln
405 410 415 Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu Ala
Thr Pro 420 425 430 Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu Gln
Ser Thr Asn Thr 435 440 445 His Thr Gln Ser Ser Ser Ser Ser Ser Asp
Gly Gly Leu Phe Arg Ser 450 455 460 Arg Pro Ala His Ser Leu Pro Pro
Gly Glu Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr Val Asp Phe Ala
Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 Glu Gln Ser
Val Val Thr Ala Pro Gly Thr Gly Gly Ser Asp Tyr Lys 500 505 510 Asp
Asp Asp Asp Lys 515 12 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 12 ccggaattca tgtctacagc ctctgcc
27 13 66 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 13 agctctagat cattagtgat ggtgatggtg atgagatcca
ccggtacctg aagtgccttg 60 tcgttt 66 14 1788 DNA Homo sapiens CDS
(7)..(1776) 14 gaattc atg tct aca gcc tct gcc gcc tcc tcc tcc tcc
tcg tct tcg 48 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser
Ser 1 5 10 gcc ggt gag atg atc gaa gcc cct tcc cag gtc ctc aac ttt
gaa gag 96 Ala Gly Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe
Glu Glu 15 20 25 30 atc gac tac aag gag atc gag gtg gaa gag gtt gtt
gga aga gga gcc 144 Ile Asp Tyr Lys Glu Ile Glu Val Glu Glu Val Val
Gly Arg Gly Ala 35 40 45 ttt gga gtt gtt tgc aaa gct aag tgg aga
gca aaa gat gtt gct att 192 Phe Gly Val Val Cys Lys Ala Lys Trp Arg
Ala Lys Asp Val Ala Ile 50 55 60 aaa caa ata gaa agt gaa tct gag
agg aaa gcg ttt att gta gag ctt 240 Lys Gln Ile Glu Ser Glu Ser Glu
Arg Lys Ala Phe Ile Val Glu Leu 65 70 75 cgg cag tta tcc cgt gtg
aac cat cct aat att gta aag ctt tat gga 288 Arg Gln Leu Ser Arg Val
Asn His Pro Asn Ile Val Lys Leu Tyr Gly 80 85 90 gcc tgc ttg aat
cca gtg tgt ctt gtg atg gaa tat gct gaa ggg ggc 336 Ala Cys Leu Asn
Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly 95 100 105 110 tct
tta tat aat gtg ctg cat ggt gct gaa cca ttg cca tat tat act 384 Ser
Leu Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr 115 120
125 gct gcc cac gca atg agt tgg tgt tta cag tgt tcc caa gga gtg gct
432 Ala Ala His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala
130 135 140 tat ctt cac agc atg caa ccc aaa gcg cta att cac agg gac
ctg aaa 480 Tyr Leu His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp
Leu Lys 145 150 155 cca cca aac tta ctg ctg gtt gca ggg ggg aca gtt
cta aaa att tgt 528 Pro Pro Asn Leu Leu Leu Val Ala Gly Gly Thr Val
Leu Lys Ile Cys 160 165 170 gat ttt ggt aca gcc tgt gac att cag aca
cac atg acc aat aac aag 576 Asp Phe Gly Thr Ala Cys Asp Ile Gln Thr
His Met Thr Asn Asn Lys 175 180 185 190 ggg agt gct gct tgg atg gca
cct gaa gtt ttt gaa ggt agt aat tac 624 Gly Ser Ala Ala Trp Met Ala
Pro Glu Val Phe Glu Gly Ser Asn Tyr 195 200 205 agt gaa aaa tgt
gac gtc ttc agc tgg ggt att att ctt tgg gaa gtg 672 Ser Glu Lys Cys
Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val 210 215 220 ata acg
cgt cgg aaa ccc ttt gat gag att ggt ggc cca gct ttc cga 720 Ile Thr
Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg 225 230 235
atc atg tgg gct gtt cat aat ggt act cga cca cca ctg ata aaa aat 768
Ile Met Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn 240
245 250 tta cct aag ccc att gag agc ctg atg act cgt tgt tgg tct aaa
gat 816 Leu Pro Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys
Asp 255 260 265 270 cct tcc cag cgc cct tca atg gag gaa att gtg aaa
ata atg act cac 864 Pro Ser Gln Arg Pro Ser Met Glu Glu Ile Val Lys
Ile Met Thr His 275 280 285 ttg atg cgg tac ttt cca gga gca gat gag
cca tta cag tat cct tgt 912 Leu Met Arg Tyr Phe Pro Gly Ala Asp Glu
Pro Leu Gln Tyr Pro Cys 290 295 300 cag tat tca gat gaa gga cag agc
aac tct gcc acc agt aca ggc tca 960 Gln Tyr Ser Asp Glu Gly Gln Ser
Asn Ser Ala Thr Ser Thr Gly Ser 305 310 315 ttc atg gac att gct tct
aca aat acg agt aac aaa agt gac act aat 1008 Phe Met Asp Ile Ala
Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn 320 325 330 atg gag caa
gtt cct gcc aca aat gat act att aag cgc tta gaa tca 1056 Met Glu
Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg Leu Glu Ser 335 340 345
350 aaa ttg ttg aaa aat cag gca aag caa cag agt gaa tct gga cgt tta
1104 Lys Leu Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu Ser Gly Arg
Leu 355 360 365 agc ttg gga gcc tcc cat ggg agc agt gtg gag agc ttg
ccc cca acc 1152 Ser Leu Gly Ala Ser His Gly Ser Ser Val Glu Ser
Leu Pro Pro Thr 370 375 380 tct gag ggc aag agg atg agt gct gac atg
tct gaa ata gaa gct agg 1200 Ser Glu Gly Lys Arg Met Ser Ala Asp
Met Ser Glu Ile Glu Ala Arg 385 390 395 atc gcc gca acc aca ggc aac
gga cag cca aga cgt aga tcc atc caa 1248 Ile Ala Ala Thr Thr Gly
Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln 400 405 410 gac ttg act gta
act gga aca gaa cct ggt cag gtg agc agt agg tca 1296 Asp Leu Thr
Val Thr Gly Thr Glu Pro Gly Gln Val Ser Ser Arg Ser 415 420 425 430
tcc agt ccc agt gtc aga atg att act acc tca gga cca acc tca gaa
1344 Ser Ser Pro Ser Val Arg Met Ile Thr Thr Ser Gly Pro Thr Ser
Glu 435 440 445 aag cca act cga agt cat cca tgg acc cct gat gat tcc
aca gat acc 1392 Lys Pro Thr Arg Ser His Pro Trp Thr Pro Asp Asp
Ser Thr Asp Thr 450 455 460 aat gga tca gat aac tcc atc cca atg gct
tat ctt aca ctg gat cac 1440 Asn Gly Ser Asp Asn Ser Ile Pro Met
Ala Tyr Leu Thr Leu Asp His 465 470 475 caa cta cag cct cta gca ccg
tgc cca aac tcc aaa gaa tct atg gca 1488 Gln Leu Gln Pro Leu Ala
Pro Cys Pro Asn Ser Lys Glu Ser Met Ala 480 485 490 gtg ttt gaa cag
cat tgt aaa atg gca caa gaa tat atg aaa gtt caa 1536 Val Phe Glu
Gln His Cys Lys Met Ala Gln Glu Tyr Met Lys Val Gln 495 500 505 510
aca gaa att gca ttg tta tta cag aga aag caa gaa cta gtt gca gaa
1584 Thr Glu Ile Ala Leu Leu Leu Gln Arg Lys Gln Glu Leu Val Ala
Glu 515 520 525 ctg gac cag gat gaa aag gac cag caa aat aca tct cgc
ctg gta cag 1632 Leu Asp Gln Asp Glu Lys Asp Gln Gln Asn Thr Ser
Arg Leu Val Gln 530 535 540 gaa cat aaa aag ctt tta gat gaa aac aaa
agc ctt tct act tac tac 1680 Glu His Lys Lys Leu Leu Asp Glu Asn
Lys Ser Leu Ser Thr Tyr Tyr 545 550 555 cag caa tgc aaa aaa caa cta
gag gtc atc aga agt cag cag cag aaa 1728 Gln Gln Cys Lys Lys Gln
Leu Glu Val Ile Arg Ser Gln Gln Gln Lys 560 565 570 cga caa ggc act
tca ggt acc ggt gga tct cat cac cat cac cat cac 1776 Arg Gln Gly
Thr Ser Gly Thr Gly Gly Ser His His His His His His 575 580 585 590
taatgatcta ga 1788 15 590 PRT Homo sapiens 15 Met Ser Thr Ala Ser
Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly 1 5 10 15 Glu Met Ile
Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp 20 25 30 Tyr
Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly 35 40
45 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln
50 55 60 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu
Arg Gln 65 70 75 80 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu
Tyr Gly Ala Cys 85 90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr
Ala Glu Gly Gly Ser Leu 100 105 110 Tyr Asn Val Leu His Gly Ala Glu
Pro Leu Pro Tyr Tyr Thr Ala Ala 115 120 125 His Ala Met Ser Trp Cys
Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu 130 135 140 His Ser Met Gln
Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn
Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe 165 170
175 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser
180 185 190 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr
Ser Glu 195 200 205 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp
Glu Val Ile Thr 210 215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly
Pro Ala Phe Arg Ile Met 225 230 235 240 Trp Ala Val His Asn Gly Thr
Arg Pro Pro Leu Ile Lys Asn Leu Pro 245 250 255 Lys Pro Ile Glu Ser
Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser 260 265 270 Gln Arg Pro
Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met 275 280 285 Arg
Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln Tyr 290 295
300 Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser Phe Met
305 310 315 320 Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr
Asn Met Glu 325 330 335 Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg
Leu Glu Ser Lys Leu 340 345 350 Leu Lys Asn Gln Ala Lys Gln Gln Ser
Glu Ser Gly Arg Leu Ser Leu 355 360 365 Gly Ala Ser His Gly Ser Ser
Val Glu Ser Leu Pro Pro Thr Ser Glu 370 375 380 Gly Lys Arg Met Ser
Ala Asp Met Ser Glu Ile Glu Ala Arg Ile Ala 385 390 395 400 Ala Thr
Thr Gly Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln Asp Leu 405 410 415
Thr Val Thr Gly Thr Glu Pro Gly Gln Val Ser Ser Arg Ser Ser Ser 420
425 430 Pro Ser Val Arg Met Ile Thr Thr Ser Gly Pro Thr Ser Glu Lys
Pro 435 440 445 Thr Arg Ser His Pro Trp Thr Pro Asp Asp Ser Thr Asp
Thr Asn Gly 450 455 460 Ser Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr
Leu Asp His Gln Leu 465 470 475 480 Gln Pro Leu Ala Pro Cys Pro Asn
Ser Lys Glu Ser Met Ala Val Phe 485 490 495 Glu Gln His Cys Lys Met
Ala Gln Glu Tyr Met Lys Val Gln Thr Glu 500 505 510 Ile Ala Leu Leu
Leu Gln Arg Lys Gln Glu Leu Val Ala Glu Leu Asp 515 520 525 Gln Asp
Glu Lys Asp Gln Gln Asn Thr Ser Arg Leu Val Gln Glu His 530 535 540
Lys Lys Leu Leu Asp Glu Asn Lys Ser Leu Ser Thr Tyr Tyr Gln Gln 545
550 555 560 Cys Lys Lys Gln Leu Glu Val Ile Arg Ser Gln Gln Gln Lys
Arg Gln 565 570 575 Gly Thr Ser Gly Thr Gly Gly Ser His His His His
His His 580 585 590 16 41 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 16 ttctgaaggg cttccaccct
ggacgaagcc acccccaccc t 41 17 69 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 17 tataagcttt
tattatttat cgtcatcgtc tttgtagtcc ggtgctgtca ccacgctctg 60 ctcgccatg
69 18 34 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 18 ccggaattcc accatggagc ttcggcagtt atcc 34 19 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 19 ccggaattcc tactgacaag gatactgt 28 20 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 20
tcttcagctg gggtattat 19 21 20 DNA Artificial Sequence Description
of Artificial Sequence Synthetic DNA 21 gctttatttc catgctgggc 20 22
27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic DNA 22 cggaattcga gctccggcag tgtcgcg 27 23 30 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
DNA 23 aactgcaggc tactgacaag gatactgtaa 30 24 31 DNA Artificial
Sequence Description of Artificial Sequence Synthetic DNA 24
ccgctcgagg aggcctcttc cgctcccggc c 31 25 35 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 25 ccgaattcct
attacggtgc tgtcaccacg ctctg 35 26 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 26 ccgctcgagg
accctatgtg gactttgctg a 31 27 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 27 ccgctcgagg
atatgtggac tttgctgagt t 31 28 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 28 ccgctcgagg
agtggacttt gctgagtttt a 31 29 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 29 ccgctcgagg
agactttgct gagttttacc g 31 30 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 30 ccgctcgagg
atttgctgag ttttaccgcc t 31 31 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 31 ccgctcgagg
agctgagttt taccgcctct g 31 32 38 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 32 ccgaattcct
attagaggcg gtaaaactca gcaaagtc 38 33 35 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 33 ccgaattcct
attaagcaaa gtccacatag ggctc 35 34 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 34 ccgaattcct
attaaaagtc cacatagggc tc 32 35 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 35 ccgaattcct
attagtccac atagggctca ac 32 36 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 36 ccgaattcct
attacacata gggctcaaca cg 32 37 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 37 ccgaattcct
attaataggg ctcaacacga cc 32 38 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 38 ccgaattcct
attagggctc aacacgaccg tc 32 39 32 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 39 ccgaattcct
attactcaac acgaccgtcc tc 32 40 16 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 40 Cys Val Glu
Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg Gly Arg Lys 1 5 10 15 41 16
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 41 Cys Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr
Asn Thr His Thr Gln 1 5 10 15 42 1568 DNA Homo sapiens CDS
(11)..(1549) 42 gaattccacc atg gac tac aag gat gac gac gac aag atg
gcg gcg cag 49 Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Ala Gln
1 5 10 agg agg agc ttg ctg cag agt gag cag cag cca agc tgg aca gat
gac 97 Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser Trp Thr Asp
Asp 15 20 25 ctg cct ctc tgc cac ctc tct ggg gtt ggc tca gcc tcc
aac cgc agc 145 Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala Ser
Asn Arg Ser 30 35 40 45 tac tct gct gat ggc aag ggc act gag agc cac
ccg cca gag gac agc 193 Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His
Pro Pro Glu Asp Ser 50 55 60 tgg ctc aag ttc agg agt gag aac aac
tgc ttc ctg tat ggg gtc ttc 241 Trp Leu Lys Phe Arg Ser Glu Asn Asn
Cys Phe Leu Tyr Gly Val Phe 65 70 75 aac ggc tat gat ggc aac cga
gtg acc aac ttc gtg gcc cag cgg ctg 289 Asn Gly Tyr Asp Gly Asn Arg
Val Thr Asn Phe Val Ala Gln Arg Leu 80 85 90 tcc gca gag ctc ctg
ctg ggc cag ctg aat gcc gag cac gcc gag gcc 337 Ser Ala Glu Leu Leu
Leu Gly Gln Leu Asn Ala Glu His Ala Glu Ala 95 100 105 gat gtg cgg
cgt gtg ctg ctg cag gcc ttc gat gtg gtg gag agg agc 385 Asp Val Arg
Arg Val Leu Leu Gln Ala Phe Asp Val Val Glu Arg Ser 110 115 120 125
ttc ctg gag tcc att gac gac gcc ttg gct gag aag gca agc ctc cag 433
Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys Ala Ser Leu Gln 130
135 140 tcg caa ttg cca gag gga gtc cct cag cac cag ctg cct cct cag
tat 481 Ser Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu Pro Pro Gln
Tyr 145 150 155 cag aag atc ctt gag aga ctc aag acg tta gag agg gaa
att tcg gga 529 Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg Glu
Ile Ser Gly 160 165 170 ggg gcc atg gcc gtt gtg gcg gtc ctt ctc aac
aac aag ctc tac gtc 577 Gly Ala Met Ala Val Val Ala Val Leu Leu Asn
Asn Lys Leu Tyr Val 175 180 185 gcc aat gtc ggt aca aac cgt gca ctt
tta tgc aaa tcg aca gtg gat 625 Ala Asn Val Gly Thr Asn Arg Ala Leu
Leu Cys Lys Ser Thr Val Asp 190 195 200 205 ggg ttg cag gtg aca cag
ctg aac gtg gac cac acc aca gag aac gag 673 Gly Leu Gln Val Thr Gln
Leu Asn Val Asp His Thr Thr Glu Asn Glu 210 215 220 gat gag ctc ttc
cgt ctt tcg cag ctg ggc ttg gat gct gga aag atc 721 Asp Glu Leu Phe
Arg Leu Ser Gln Leu Gly Leu Asp Ala Gly Lys Ile 225 230 235 aag cag
gtg ggg atc atc tgt ggg cag gag agc acc cgg cgg atc ggg 769 Lys Gln
Val Gly Ile Ile Cys Gly Gln Glu Ser Thr Arg Arg Ile Gly 240 245 250
gat tac aag gtt aaa tat ggc tac acg gac att gac ctt ctc agc gct 817
Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp Leu Leu Ser Ala 255
260 265 gcc aag tcc aaa cca atc atc gca gag cca gaa atc cat ggg gca
cag 865 Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile His Gly Ala
Gln 270 275 280 285 ccg ctg gat ggg gtg acg ggc ttc ttg gtg ctg atg
tcg gag ggg ttg 913 Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met
Ser Glu Gly Leu 290 295 300 tac aag gcc cta gag gca gcc cat ggg cct
ggg cag gcc aac cag gag 961 Tyr Lys Ala Leu Glu Ala Ala His Gly Pro
Gly Gln Ala Asn Gln Glu 305 310 315 att gct gcg atg att gac act gag
ttt gcc aag cag acc tcc ctg gac 1009 Ile Ala Ala Met Ile Asp Thr
Glu Phe Ala Lys Gln Thr Ser Leu Asp 320 325 330 gca gtg gcc cag gcc
gtc gtg gac cgg gtg aag cgc atc cac agc gac 1057 Ala Val Ala Gln
Ala Val Val Asp Arg Val Lys Arg Ile His Ser Asp 335 340 345 acc ttc
gcc agt ggt ggg gag cgt gcc agg ttc tgc ccc cgg cac gag 1105 Thr
Phe Ala Ser Gly Gly Glu Arg Ala Arg Phe Cys Pro Arg His Glu 350 355
360 365 gac atg acc ctg cta gtg agg aac ttt ggc tac ccg ctg ggc caa
atg 1153 Asp Met Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro Leu Gly
Gln Met
370 375 380 agc cag ccc aca ccg agc cca gcc cca gct gca gga gga cga
gtg tac 1201 Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly Gly
Arg Val Tyr 385 390 395 cct gtg tct gtg cca tac tcc agc gcc cag agc
acc agc aag acc agc 1249 Pro Val Ser Val Pro Tyr Ser Ser Ala Gln
Ser Thr Ser Lys Thr Ser 400 405 410 gtg acc ctc tcc ctt gtc atg ccc
tcc cag ggc cag atg gtc aac ggg 1297 Val Thr Leu Ser Leu Val Met
Pro Ser Gln Gly Gln Met Val Asn Gly 415 420 425 gct cac agt gct tcc
acc ctg gac gaa gcc acc ccc acc ctc acc aac 1345 Ala His Ser Ala
Ser Thr Leu Asp Glu Ala Thr Pro Thr Leu Thr Asn 430 435 440 445 caa
agc ccg acc tta acc ctg cag tcc acc aac acg cac acg cag agc 1393
Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr His Thr Gln Ser 450
455 460 agc agc tcc agc tct gac gga ggc ctc ttc cgc tcc cgg ccc gcc
cac 1441 Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser Arg Pro
Ala His 465 470 475 tcg ctc ccg cct ggc gag gac ggt cgt gtt gag ccc
tat gtg gac ttt 1489 Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu
Pro Tyr Val Asp Phe 480 485 490 gct gag ttt tac cgc ctc tgg agc gtg
gac cat ggc gag cag agc gtg 1537 Ala Glu Phe Tyr Arg Leu Trp Ser
Val Asp His Gly Glu Gln Ser Val 495 500 505 gtg aca gca ccg
tgatgagcgg ccgcatcgt 1568 Val Thr Ala Pro 510 43 513 PRT Homo
sapiens 43 Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Ala Gln Arg
Arg Ser 1 5 10 15 Leu Leu Gln Ser Glu Gln Gln Pro Ser Trp Thr Asp
Asp Leu Pro Leu 20 25 30 Cys His Leu Ser Gly Val Gly Ser Ala Ser
Asn Arg Ser Tyr Ser Ala 35 40 45 Asp Gly Lys Gly Thr Glu Ser His
Pro Pro Glu Asp Ser Trp Leu Lys 50 55 60 Phe Arg Ser Glu Asn Asn
Cys Phe Leu Tyr Gly Val Phe Asn Gly Tyr 65 70 75 80 Asp Gly Asn Arg
Val Thr Asn Phe Val Ala Gln Arg Leu Ser Ala Glu 85 90 95 Leu Leu
Leu Gly Gln Leu Asn Ala Glu His Ala Glu Ala Asp Val Arg 100 105 110
Arg Val Leu Leu Gln Ala Phe Asp Val Val Glu Arg Ser Phe Leu Glu 115
120 125 Ser Ile Asp Asp Ala Leu Ala Glu Lys Ala Ser Leu Gln Ser Gln
Leu 130 135 140 Pro Glu Gly Val Pro Gln His Gln Leu Pro Pro Gln Tyr
Gln Lys Ile 145 150 155 160 Leu Glu Arg Leu Lys Thr Leu Glu Arg Glu
Ile Ser Gly Gly Ala Met 165 170 175 Ala Val Val Ala Val Leu Leu Asn
Asn Lys Leu Tyr Val Ala Asn Val 180 185 190 Gly Thr Asn Arg Ala Leu
Leu Cys Lys Ser Thr Val Asp Gly Leu Gln 195 200 205 Val Thr Gln Leu
Asn Val Asp His Thr Thr Glu Asn Glu Asp Glu Leu 210 215 220 Phe Arg
Leu Ser Gln Leu Gly Leu Asp Ala Gly Lys Ile Lys Gln Val 225 230 235
240 Gly Ile Ile Cys Gly Gln Glu Ser Thr Arg Arg Ile Gly Asp Tyr Lys
245 250 255 Val Lys Tyr Gly Tyr Thr Asp Ile Asp Leu Leu Ser Ala Ala
Lys Ser 260 265 270 Lys Pro Ile Ile Ala Glu Pro Glu Ile His Gly Ala
Gln Pro Leu Asp 275 280 285 Gly Val Thr Gly Phe Leu Val Leu Met Ser
Glu Gly Leu Tyr Lys Ala 290 295 300 Leu Glu Ala Ala His Gly Pro Gly
Gln Ala Asn Gln Glu Ile Ala Ala 305 310 315 320 Met Ile Asp Thr Glu
Phe Ala Lys Gln Thr Ser Leu Asp Ala Val Ala 325 330 335 Gln Ala Val
Val Asp Arg Val Lys Arg Ile His Ser Asp Thr Phe Ala 340 345 350 Ser
Gly Gly Glu Arg Ala Arg Phe Cys Pro Arg His Glu Asp Met Thr 355 360
365 Leu Leu Val Arg Asn Phe Gly Tyr Pro Leu Gly Gln Met Ser Gln Pro
370 375 380 Thr Pro Ser Pro Ala Pro Ala Ala Gly Gly Arg Val Tyr Pro
Val Ser 385 390 395 400 Val Pro Tyr Ser Ser Ala Gln Ser Thr Ser Lys
Thr Ser Val Thr Leu 405 410 415 Ser Leu Val Met Pro Ser Gln Gly Gln
Met Val Asn Gly Ala His Ser 420 425 430 Ala Ser Thr Leu Asp Glu Ala
Thr Pro Thr Leu Thr Asn Gln Ser Pro 435 440 445 Thr Leu Thr Leu Gln
Ser Thr Asn Thr His Thr Gln Ser Ser Ser Ser 450 455 460 Ser Ser Asp
Gly Gly Leu Phe Arg Ser Arg Pro Ala His Ser Leu Pro 465 470 475 480
Pro Gly Glu Asp Gly Arg Val Glu Pro Tyr Val Asp Phe Ala Glu Phe 485
490 495 Tyr Arg Leu Trp Ser Val Asp His Gly Glu Gln Ser Val Val Thr
Ala 500 505 510 Pro 44 51 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 44 cccgaattcc accatggact
acaaggatga cgacgacaag atggcggcgc a 51 45 35 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 45 gatgcggccg
ctcatcacgg tgctgtcacc acgct 35 46 34 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 46 ccgctcgagg
acggcccgcc cactcgctcc cgcc 34 47 34 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 47 ccgctcgagg
actcccgcct ggcgaggacg gtcg 34 48 34 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 48 ccgctcgagg
agacggtcgt gttgagccct atgt 34
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