U.S. patent application number 11/651111 was filed with the patent office on 2007-10-18 for wt1 interacting protein wtip.
This patent application is currently assigned to Haruo SUGIYAMA. Invention is credited to Eui Ho Kim, Haruo Sugiyama.
Application Number | 20070243611 11/651111 |
Document ID | / |
Family ID | 18542325 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070243611 |
Kind Code |
A1 |
Sugiyama; Haruo ; et
al. |
October 18, 2007 |
WT1 interacting protein WTIP
Abstract
A human WT1 interacting protein (WTIP) having the amino acid
sequence as set forth in SEQ ID NO: 2 or a gene encoding it.
Inventors: |
Sugiyama; Haruo; (Osaka,
JP) ; Kim; Eui Ho; (Osaka, JP) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Haruo SUGIYAMA
|
Family ID: |
18542325 |
Appl. No.: |
11/651111 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10181804 |
Jul 23, 2002 |
7173111 |
|
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PCT/JP01/00461 |
Jan 24, 2001 |
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11651111 |
Jan 9, 2007 |
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Current U.S.
Class: |
435/358 ;
435/252.33; 435/254.21; 435/254.3; 435/320.1; 435/414; 536/22.1;
536/24.1 |
Current CPC
Class: |
C07K 14/4702
20130101 |
Class at
Publication: |
435/358 ;
435/252.33; 435/254.21; 435/254.3; 435/320.1; 435/414; 536/022.1;
536/024.1 |
International
Class: |
C12N 5/16 20060101
C12N005/16; C07H 21/00 20060101 C07H021/00; C07H 21/04 20060101
C07H021/04; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101
C12N001/19; C12N 1/21 20060101 C12N001/21; C12N 15/63 20060101
C12N015/63; C12N 5/14 20060101 C12N005/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2000 |
JP |
2000-14949 |
Claims
1-18. (canceled)
19. A DNA that specifically hybridizes to a DNA comprising the base
sequence as set forth in SEQ ID NO: 1 and that has a chain length
of at least 15 bases.
20. A nucleic acid encoding a WT1 interacting protein having the
amino acid sequence as set forth in SEQ ID NO: 2, or a protein that
has an amino acid sequence in which one or a plurality of amino
acids have been substituted, deleted, inserted, and/or added in the
amino acids of said protein and that is functionally equivalent to
the protein having the amino acid sequence as set forth in SEQ ID
NO: 2.
21. A vector comprising the nucleic acid according to claim 20.
22. A host cell carrying the vector according to claim 21.
23. A DNA hybridizing under stringent conditions to DNA having the
base sequence as set forth in SEQ ID NO: 1 and that is functionally
equivalent to the protein having the amino acid sequence as set
forth in SEQ ID NO: 2.
24. A vector comprising the DNA according to claim 20.
25. A host cell carrying the vector according to claim 21.
26. A nucleic acid encoding a WT1 interacting protein comprising an
amino acid sequence from Glu at position 449 to Met at position 541
in SEQ ID NO: 2.
27. A vector comprising the nucleic acid encoding the protein
according to claim 26.
28. A host cell carrying the vector according to claim 27.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 10/181,804, filed Jul. 23, 2002, which is the national stage
application of PCT/JP01/00461, filed Jan. 24, 2001, which claims
the priority to Japan Application No. 2000-014949, filed Jan. 24,
2000, which are all incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a protein (WT1 interacting
protein) (WTIP) that interacts with the WTI protein, a gene
encoding the same, and uses thereof.
BACKGROUND OF THE INVENTION
[0003] WT1 (Wilms' tumor genie 1) gene is a transcription
regulatory factor (Call K. M. et al., Cell 60, 509-520, 1990;
Gessler M. et al., Nature 343, 774-778, 1990) discovered in the
course of identifying a causative gene of Wilms' tumor which is a
pediatric kidney tumor, and functions as a tumor suppressor gene in
at least some Wilms' tumors. It was also discovered that its level
of expression is high in most leukemia cells, and it is becoming
clear that its level of expression at the first medical examination
for leukemia correlates well with prognosis, and it is extremely
useful as a marker of minimal residual disease (MRD) of leukemia
(Inoue K. et al., Blood 84, 3071-3079, 1994).
[0004] In normal tissue, WT1 is highly expressed in the testis, the
ovary, the spleen, the mesenchymal mesothelium as well as in the
embryonal kidney. Among malignant tumors, it is also highly
expressed in leukemia, malignant mesothelioma, and solid tumors
such as lung cancer. There is increasing evidence that the WT1 gene
is a gene having a variety of functions in organogenesis,
oncogenesis and the like (Reddy J. C. et al., Biochim. Biophys.
Acta. 1287, 1-28, 1996; Davices R. et al., Cancer Res. 59,
1747-1751, 1999).
[0005] The WT1 gene is mainly translated into four proteins by
alternative splicing in the exons (A in FIG. 1). The longest gene
product, in which exon 5 comprising 17 amino acid residues (17AA)
and three amino acid residues (KTS.sup.1) in between the third and
the fourth Zinc fingers have been inserted, is designated herein as
WT1(+/+).
[0006] It has been demonstrated that the KTS-containing WT1(+/+)
has a weak DNA binding ability and binds to the mRNA splicing
protein, whereas the WT1(+/-) having a potent DNA-binding ability
functions as a transcription regulatory factor.
[0007] The WT1 protein is roughly composed of two regions, i.e.,
the function regulatory region and the DNA-binding region
containing zinc fingers. In the function regulatory region, as
shown in A in FIG. 1, have two domains that suppress or activate
transcription. The assumption that proteins that bind to these
regions may be responsible for the regulation of functions led to
the discovery of various WT1 interacting proteins. They include
various proteins such as p53 (Malheswran S. et al., Proc. Natl.
Acad. Sci. USA 90, 5100-5104, 1993), ubiquitine conjugating enzyme
9 (Wany Z. Y. et al., J. Biol. Chem. 271, 24811-24816, 1996).,
par-4 (Johnstone R. W. et al., Mol. Cell. Biol. 16, 6945-6956,
1990), U2AF65 (Ravis R. C. et al., Geves. Rev. 12, 3217-3225,
1998), and hsp70 (Maheswaran S. et al., Geves Rev. 12, 1108-1120,
1998). However, there are no reports of specific binding proteins
in leukemia cells.
DISCLOSURE OF THE INVENTION
[0008] Thus, it is an object of the present invention to provide a
novel protein that interacts with (binds to) the WT1 protein, a
gene encoding it, uses thereof and the like.
[0009] Thus, the present invention provides (1) a WT1 interacting
protein having the amino acid sequence as set forth in SEQ ID NO:
2, or a protein that has an amino acid sequence in which one or a
plurality of amino acids have been substituted, deleted, inserted,
and/or added in the amino acids of said protein and that is
functionally equivalent to the protein having the amino acid
sequence as set forth in SEQ ID NO: 2.
[0010] The present invention also provides (2) a protein that is
encoded by DNA hybridizing under a stringent condition to DNA
having the nucleotide sequence as set forth in SEQ ID NO: 1 and
that is functionally equivalent to the protein having the amino
acid sequence as set forth in SEQ ID NO: 2.
[0011] The present invention also provides (3) a WT1 interacting
protein comprising an amino acid sequence from positions 449 to 541
in SEQ ID NO: 2.
[0012] The present invention also provides (4) a function modulator
of the WT1 protein comprising, as an active ingredient, a
polypeptide comprising an amino acid sequence from Glu at position
449 to Met at position 541 in SEQ ID NO: 2.
[0013] The present invention also provides (5) a partial peptide of
the protein described in the above (1) or (2).
[0014] The present invention also provides (6) a gene encoding a
protein described in any of the above (1) to (3).
[0015] The present invention also provides (7) a vector comprising
the gene described in the above (6).
[0016] The present invention also provides (8) a host cell carrying
the vector described in the above (7).
[0017] The present invention also provides (9) a method of
producing a protein described in any of the above (1) to (3), said
method comprising culturing the host cell of the above (8).
[0018] The present invention also provides (10) an antibody to a
protein described in any of the above (1) to (3).
[0019] The present invention also provides (11) a method of
detecting or determining a protein described in any of the above
(1) to (3) comprising:
[0020] (a) bringing the antibody described in the above (10) into
contact with a sample expected to contain said protein; and
[0021] (b) detecting or determining the formation of an immune
complex between said antibody and said protein or immunostaining
the cells that express said protein using said antibody.
[0022] The present invention also provides (12) DNA that
specifically hybridizes to DNA comprising the base sequence as set
forth in SEQ ID NO: 1 and that has a chain length of at least 15
bases.
[0023] The present invention also provides (13) a method of
screening a compound that binds to a protein described in any of
the above (1) to (3), said method comprising the steps of:
[0024] (a) bringing a sample to be tested into contact with said
protein or a partial peptide thereof;
[0025] (b) detecting the binding activity of said sample with said
protein or a partial peptide thereof; and
[0026] (c) selecting a compound having an activity of binding to
said protein or a partial peptide thereof.
[0027] The present invention also provides (14) a compound that can
be isolated by the method described in the above (13) and that
binds to a protein described in any of the above (1) to (3).
[0028] The present invention also provides (15) a method of
screening a compound that promotes or inhibits the activity of a
protein described in any of the above (1) to (3), said method
comprising the steps of:
[0029] (a) culturing the cells that express said protein in the
presence of a sample to be tested;
[0030] (b) detecting the growth of said cells; and
[0031] (c) selecting a compound that promotes or inhibits said
growth as compared to when detected in the absence of said sample
to be tested.
[0032] The present invention also provides (16) a compound that can
be isolated by the method described in the above (15) and that
promotes or inhibits the activity of a protein described in any of
the above (1) to (3).
[0033] The present invention also provides (17) a method of
screening a compound that promotes or inhibits the binding of the
WT1 interacting protein described in any of the above (1) to (3)
with the WT1 protein, said method comprising the steps of:
[0034] (a) allowing the WT1 interacting protein to react with the
WT1 protein in the presence of a sample to be tested;
[0035] (b) determining the binding activity of both proteins;
and
[0036] (c) selecting a compound that promotes or inhibits said
binding as compared to when detected in the absence of said sample
to be tested.
[0037] The present invention also provides (18) a compound that can
be isolated by the method described in the above (17) and that
promotes or inhibits the binding of the WT1 interacting protein
described in any of the above (1) to (3) with the WT1 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In FIG. 1, A is a drawing that shows the structure of cDNA
encoding the full-length of the WT1 protein, whereas B is a drawing
that shows the structure of a fusion protein (GST-WT2) of the
suppressive domain of the WT1 protein and GST (glutathione
S-transferase) and a fusion protein (GST-WT3) of the suppressive
domain and the activating domain and GST, and a fusion protein
(GST-WT4) of the activating domain and GST.
[0039] FIG. 2 is an electrophoretogram that shows the reactivity of
the extracts of the K562 cells with the fusion protein shown in B
of FIG. 1 and with GST alone. It shows that there is an about 115
kDa protein present in the extract of the K562 cells, that binds to
the suppressive domain of the WT1 protein.
[0040] FIG. 3 is a drawing that shows an elution profile wherein
the cell-free extracts (the centrifugation supernatant after the
sonic disruption of the cells) of the K562 cells were separated by
an anion exchange chromatography with HiLoad 16/10 Q Sepharose High
Performance. Activity was observed in fraction Nos. 26-35 by the
west western blot method.
[0041] FIG. 4 is a drawing that shows an elution profile wherein
the active fractions obtained in FIG. 3 were separated by an anion
exchange chromatography with MONO Q HR 5/5. Activity was observed
in fraction Nos. 14 to 17 by the west western blot method.
[0042] FIG. 5 is a drawing that shows an elution profile wherein
the active fractions obtained in FIG. 5 were separated by a
hydrophobic chromatography with Phenyl Superrose HR 5/5. Activity
was observed in fraction Nos. 13 to 14 by the west western blot
method.
[0043] FIG. 6 is an electrophoretogram that shows the result of
protein detection with Coomassie brilliant blue (CBB), and the west
western blot method detection by the reactivity with GST-WT3,
GST-WT2 and GST after the active fractions obtained in FIG. 5
containing 115 kDa protein were transferred to a PVDF membrane. It
can be seen that the 115 kDa protein binds to the suppressive
domain of WT1.
[0044] In FIG. 7, A shows the structure of the full-length WTIP, B
shows the structure of polypeptides (WTIP 1-406 and WTIP 1-178)
containing the WW domain at the N-terminal end of WTIP and a
polypeptide (WTIP 1-98) containing no WW domains in which a
histidine (H is) tag (tag) has been attached to the C-terminal of
each polypeptide.
[0045] FIG. 8 is an electrophoretogram that shows the result of
detection by the reactivity with anti-His tagged polypeptide
antibody, GST-WT3, and GST after the polypeptide shown in FIG. 7
was electrophoresed. It can be seen that WTIP 1-406 and WTIP 1-178
containing the WW domain react, but that WTIP 1-98, that does not
contain the WW domain, does not react, with GST-WT3 that contains
the suppressive domain, and GST that does not contain the
suppressive domain does not react with any of the WTIP fragments,
indicating that the WW domain in WTIP and a part of the suppressive
domain of the WT1 protein react.
[0046] FIG. 9 is an electrophoretogram that shows that WT1 and WTIP
bind in the cell.
[0047] In order to obtain a novel protein that binds to the WT1
protein, WT1 interacting proteins were searched for using a human
leukemia cell line K562 (Yamagami T. et al., Blood 87, 2878-2885,
1996) in which growth inhibition is observed when using an
antisense oligo DNA of WT1. In a west western blot using a fusion
protein of the WT1 regulatory region and glutathione S-transferase
(GST), an intracellular protein that binds to the fusion protein
was noted, which thereby was separated by a column chromatography
and SDS-PAGE, and a gene was identified by peptide mapping and by
screening with expression. As a result, cDNA of a novel gene WT1
interacting protein (WTIP) was cloned.
[0048] Thus the present invention provides a WT1 interacting
protein having the amino acid sequence as set forth in SEQ ID NO:
1.
[0049] In the sequence described in SEQ ID NO: 1, the nucleotide
sequence of positions 1 to 1358 corresponds to FBP11 (Chan D. C. et
al., EMBO J. 15, 1045-1054, 1996) which is one of the proteins
(formin binding protein) binding to formin involved in the
development of mouse legs and kidney and HYPA (Huntington yeast
partner A) (Faber P. W. et al., Hum. Mol. Genet. 7, 1463-1467,
1998) which is one of the proteins binding to Huntington that is
considered to be a causative gene of Huntington's chorea, a human
neurodegenerative disease, and the amino acid sequence
corresponding to the nucleotide sequence of positions 1636 to 2980
in SEQ ID NO: 1 corresponds to NY-REN-6 (Scanlan M. J. et al., Int.
J. Cancer 83, 456-464, 1999) identified as an autoantigen that
recognizes an autoantibody in the serum of human patients with
kidney cancer.
[0050] Thus, a protein having a full-length and partial amino acid
sequence, comprising the amino acid sequence corresponding to the
nucleotide sequence of positions 1359 to 1635 in SEQ ID NO: 1, is
novel. Thus, the present invention provides a protein comprising an
amino acid sequence that corresponds to the nucleotide sequence of
positions 1359 to 1635 in SEQ ID NO: 1.
[0051] The present invention also provides a gene, for example DNA
or RNA, encoding the above protein. Said DNA is specifically
cDNA.
[0052] The present invention also provides a vector comprising the
above DNA, for example an expression vector.
[0053] In accordance with the present invention, it was found that
the WW domain from Gly at position 141 to Asp at position 217 in
SEQ ID NO: 2 is a binding region with the WT1 protein. Thus the
present invention provides a regulatory agent of WT1 protein
function comprising, as an active ingredient, a polypeptide
comprising an amino acid sequence from Gly at position 141 to Asp
at position 217 in SEQ ID NO: 2. In accordance with the present
invention, a polypeptide having an amino acid sequence of position
1 (Met) to position 98 (Gly) in SEQ ID NO: 2 does not bind to the
WT1 protein, but a polypeptide having an amino acid sequence of
position 1 (Met) to position 178 (Ala) binds to the WT1 protein.
Thus, according to one aspect of the present invention, there is
provided a regulatory agent of WT1 protein function comprising as
an active ingredient a polypeptide having or comprising an amino
acid sequence from position 99 (Gln) to position 178 (Ala) in SEQ
ID NO: 2.
[0054] In accordance with the present invention, a polypeptide
having an amino acid sequence of position 1 (Met) to position 406
(Glu) in SEQ ID NO: 2 binds to the WT1 protein. Thus, according to
one aspect of the present invention, there is provided a regulatory
agent of WT1 protein function comprising, as an active ingredient,
a polypeptide having or comprising an amino acid sequence from
position 99 (Gln) to position 406 (Glu) in SEQ ID NO: 2. More
generally, the present invention provides a regulatory agent of WT1
protein function comprising, as an active ingredient, a polypeptide
or a protein from any amino acid of position 1 (Met) to position 99
(Gin) to any amino acid of position 189 (Glu) to position 406 (Glu)
or position 957 (Gln) in SEQ ID NO: 2.
[0055] The isolation of WTIP and the cloning of cDNA encoding it is
described in Example 1. In the example, human leukemia cell-like
K562 (Japanese Cancer Research Resource Banks) was used as a source
for isolating WTIP and DNA encoding it, but other cells that
produce WTIP may also be used.
[0056] The present invention encompasses proteins functionally
equivalent to the WT1 interacting protein. Such proteins include
homolog proteins of other organisms corresponding to the human WT1
interacting protein and mutants of the human WT1 interacting
protein. As used herein the term "functionally equivalent" means
that the subject protein has a biological activity equivalent to
the above WT1 interacting protein. The biological activity is, for
example, a binding activity with the WT1 protein.
[0057] As a method well known to a person skilled in the art for
preparing a protein functionally equivalent to a certain protein,
there is known a method of introducing mutation in the protein. For
example, a person skilled in the art can use site-directed
mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275;
Zoller, M. J. and Smith, M. (1983) Methods Enzymol. 100, 468-500;
Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer
W. and Fritz H. J. (1987) Methods Enzymol. 154, 350-367; Kunkel, T.
A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492; Kunkel (1988)
Methods Enzymol. 85, 2763-2766) to introduce as appropriate a
mutation in amino acids of the human WT1 interacting protein in
order to prepare a protein functionally equivalent to the human WTI
interacting protein.
[0058] Mutation in amino acids also occurs spontaneously in nature.
Thus, proteins that have an amino acid sequence in which one or a
plurality of amino acids are mutated in the amino acid sequence of
the human WTI interacting protein and that are functionally
equivalent the human WT1 interacting protein are also encompassed
in the present invention.
[0059] As proteins functionally equivalent to the WT1 interacting
protein of the present invention, there can be specifically
mentioned those in which one or more than one, preferably two or
more and 30 or less, more preferably two or more and 10 or less
amino acids have been deleted in the amino acid sequence as set
forth in SEQ ID NO: 2, those in which one or more than one,
preferably two or more and 30 or less, more preferably two or more
and 10 or less amino acids have been added in the amino acid
sequence as set forth in SEQ ID NO: 2, and those in which one or
more than one, preferably two or more and 30 or less, more
preferably two or more and 10 or less amino acids have been
substituted with other amino acids in the amino acid sequence as
set forth in SEQ ID NO: 2.
[0060] For the amino acid residue that is mutated, it is preferably
mutated to another amino acid that retains the property of the
amino acid side chain. For example, as the properties of amino acid
side chains, there may be mentioned hydrophobic amino acids (A, I,
L, M, F, P, W, Y, v), hydrophilic amino acids (R, D, N, C, E, Q, G,
H, K, S, T), amino acids having an aliphatic side chain (G, A, V,
L, I, P), amino acids having a hydroxy group-containing side chain
(S, T, Y), amino acids having a sulfur-containing side chain (C,
M), amino acids having a carboxylic acid- or amide-containing side
chain (D, N, E, Q), amino acids having a base-containing side chain
(R, K, H), and amino acids having an aromatic-containing side chain
(H, F, Y, W) (all letters in parentheses indicate a one-letter
representation of amino acids).
[0061] It is already known that proteins having an amino acid
sequence that has been modified by deletion, addition of one or a
plurality of amino acid residues and/or substitution with another
amino acid in a certain amino acid sequence can retain the
biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA
(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.
USA (1982) 79, 6409-6413).
[0062] As a protein in which one or a plurality of amino acids have
been added to the amino acid sequence (SEQ ID NO: 2) of the human
WT1 interacting protein, there can be mentioned a fusion protein
that contains the human WT1 interacting protein. A fusion protein
is a fusion of the human WT1 interacting protein and another
peptide or protein, and is included in the present invention. In a
method of creating a fusion protein, DNA encoding the human WTI
interacting protein of the present invention and DNA encoding
another peptide or protein are linked in a frame with each other,
which is then introduced into an expression vector and expressed in
a host cell, and a method well known to a person skilled in the art
can be used. Any other peptide or protein that is subjected to
fusion with the protein of the present invention is not
specifically limited.
[0063] As another peptide that is subjected to fusion with the
protein of the present invention, for example, known peptides may
be used including FLAG (Hopp, T. P. et al., BioTechnology (1988) 6,
1204-1210), 6 x His (SEQ ID NO: 13) comprising 6 H is (histidine)
(SEQ ID NO: 13) residues, 10 x His (SEQ ID NO: 14), influenza
hemagglutinin (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 .alpha.-tubulin, B-tag, fragments of
Protein C, and the like. As another protein that is subjected to
fusion with the protein of the present invention, there may be
mentioned, for example, GST (glutathione S-transferase), HA
(influenza agglutinin), the constant regions of immunoglobulin,
.beta.-galactosidase, MBP (maltose-binding protein), and the
like.
[0064] DNA encoding such a peptide or protein is fused to DNA
encoding the protein of the present invention, and the fused DNA
thus prepared is expressed to prepare the fusion protein.
[0065] As a method well known to a person skilled in the art for
preparing a protein functionally equivalent to a certain protein,
there can be mentioned a method that employs the hybridization
technology (Sambrook, J. et al., Molecular Cloning 2nd ed.,
9.47-9.58, Cold Spring Harbor Laboratory Press, 1989).
[0066] Thus, it can be routinely performed by a person skilled in
the art that a DNA sequence (SEQ ID NO: 1) encoding the human WT1
interacting protein or a portion thereof is used to isolate DNA
having a high homology therewith, and from said DNA a protein
functionally equivalent to the human WTI interacting protein can be
routinely isolated. Thus, a protein encoded by DNA that hybridizes
to DNA encoding the human WT1 interacting protein or DNA comprising
a portion thereof and that is functionally equivalent to the human
WT1 interacting protein is also included in the present
invention.
[0067] As such a protein, there can be mentioned a homolog from a
mammal other than the human (for example, a protein encoded by a
monkey, mouse, rat, rabbit, and bovine gene). When a cDNA having a
high homology with DNA encoding the human WT1 interacting protein
is isolated from an animal, preferably tissues such as the heart,
the placenta, the testis and the like are used.
[0068] A hybridization condition for isolating a DNA encoding a
protein functionally equivalent to the human WT1 interacting
protein may be selected, as appropriate, by a person skilled in the
art. As the hybridization condition, there can be mentioned a low
stringent condition. A low stringent condition is, for example,
42.degree. C., 2.times.SSC, and 0.1% SDS, and preferably 50.degree.
C., 2.times.SSC, and 0.1% SDS.
[0069] More preferably, there can be mentioned a high stringent
condition. As a high stringent condition, for example, there can be
mentioned 65.degree. C., 2.times.SSC, and 0.1% SDS. In these
conditions, the higher the temperature is the higher the homology
of the DNA obtained is. However, as elements affecting the
stringency of hybridization, a plurality of elements can be
conceived such as a salt concentration in addition to temperature,
and a person skilled in the art can select, as appropriate, these
elements to attain a similar stringency.
[0070] Isolation can also be attained by utilizing a gene
amplifying method using primers synthesized based on the sequence
information of DNA (SEQ ID NO: 1) encoding the human WT1
interacting protein, for example a polymerase chain reaction (PCR),
in place of hybridization.
[0071] A protein functionally equivalent to the human WTI
interacting protein encoded by the DNA isolated by such a
hybridization technology or gene amplification technology usually
has a high homology with the human WT1 interacting protein in terms
of the amino acid sequence.
[0072] The protein of the present invention also includes a protein
that is functionally equivalent to the human WT1 interacting
protein and that has a high homology with the amino acid sequence
as set forth in SEQ ID NO: 2. A high homology as used herein means
a homology of 70% or higher, preferably 80% or higher, more
preferably 90% or higher, and more preferably 95% or higher in
identity.
[0073] In order to determine the homology of proteins, an algorithm
described in an article (Wilbur, W. J. and Lipman, D. J., Proc.
Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.
[0074] The protein of the present invention may differ in amino
acid sequence, molecular weight, isoelectric point, or the presence
or absence and shapes of sugar chains depending on the cells,
described below, that produce it, the host cells, and the method of
purification. However, it is included in the present invention as
long as the protein obtained retains functions equivalent to the
human WT1 interacting protein of the present invention (SEQ ID NO:
2). For example, when the protein of the present invention is
expressed in prokaryotic cells such as Escherichia coli (E. coli),
a methionine residue is added to the N-terminal of the amino acid
sequence of the original protein. Also, when it is expressed in
eukaryotic cells such as mammalian cells, the signal sequence at
the N-terminal is removed. The protein of the present invention
encompasses such proteins.
[0075] The protein of the present invention can be prepared by a
person skilled in the art in a known method either recombinantly or
as natural protein. In the case of recombinant proteins, DNA (such
as DNA having the base sequence as set forth in SEQ ID NO: 1)
encoding a protein of the present invention is integrated into a
suitable expression vector, which in turn is introduced into a
suitable host cell and a recombinant is recovered therefrom to
obtain an extract. Then the extract is purified by subjecting it to
ion exchange, reverse-phase, gel filtration chromatography, or
affinity chromatography in which an antibody against the protein of
the present invention has been immobilized on the column, or by
using a combination of a plurality of columns to prepare the
protein of the present invention.
[0076] When the protein of the present invention was expressed as a
fusion protein with the glutathione S-transferase protein or as a
recombinant protein to which a plurality of histidines were added
in a host cell (for example, in an animal cell or E. coli), the
expressed recombinant protein can be purified by a glutathione
column or a nickel column.
[0077] After purification of the fusion protein, the regions other
than the protein of interest among the fusion protein may be
removed, as desired, by cleaving with thrombin, Factor Xa etc. In
the case of a natural protein, it can be purified and isolated by
subjecting an extract of a tissue or cell that expresses the
protein of the present invention it to an affinity column to which
an antibody, described below, that binds to the WT1 interacting
protein is bound. The antibody may be a polyclonal antibody or a
monoclonal antibody.
[0078] The present invention also encompasses the partial peptides
of the protein of the present invention. The partial peptides
comprising the amino acid sequence specific to the protein of the
present invention comprises at least 7 amino acids, preferably 8
amino acids or more, and more preferably 9 amino acids or more.
Said partial peptides can be used for preparing an antibody against
the protein of the present invention, screening compounds that bind
to the protein of the present invention, and screening promoters
and inhibitors of the protein of the present invention.
Furthermore, they can be an antagonist to ligands of the protein of
the present invention.
[0079] As the partial peptides of the present invention, for
example, there can be mentioned partial peptides comprising an
active center of the protein comprising the amino acid sequence as
set forth in SEQ ID NO: 2. There can also be mentioned partial
peptides containing one or a plurality of regions of the
hydrophobic regions and hydrophilic regions deduced from the
hydrophobicity plot analysis. These partial peptides may contain
part or all of a hydrophobic region, or contain part or all of a
hydrophilic region.
[0080] The partial peptide of the present invention can be produced
by gene engineering technology, known peptide synthetic methods, or
by cleaving the protein of the present invention with a suitable
peptidase. As the peptide synthesis method, any of a solid-phase
synthesis and a liquid-phase synthesis can be used.
[0081] The present invention also relates to DNA encoding the
protein of the present invention. The DNA of the present invention
can be used for the in vivo or in vitro production of the protein
of the present invention and, besides, the application in gene
therapy to diseases associated with aberrations in the gene
encoding the protein of the present invention is contemplated.
[0082] The DNA of the present invention may be in any form as long
as it can encode the protein of the present invention. Thus, it can
be any of cDNA synthesized from mRNA, genomic DNA, or chemically
synthesized DNA. Furthermore, it contains DNA having any base
sequence based on the degeneracy of genetic code, as long as it
encodes the protein of the present invention.
[0083] The DNA of the present invention can be prepared by any
method known to a person skilled in the art. For example, it can be
prepared by preparing a cDNA library from the cells expressing the
protein of the present invention and subjecting it to hybridization
using, as the probe, part of the DNA sequence (for example SEQ ID
NO: 1) of the present invention. CDNA libraries may be prepared by
a method described in Sambrook, J. et al., Molecular Cloning, Cold
Spring Harbor Laboratory Press (1989), or commercially available
DNA libraries may be used. It is also possible to prepare RNA from
the cells expressing the protein of the present invention,
synthesizing an oligo DNA based on the DNA sequence (for example
SEQ ID NO: 1) of the present invention, which is then used as a
primer in a PCR reaction to amplify cDNA encoding the protein of
the present invention.
[0084] By determining the nucleotide sequence of the cDNA obtained,
the translation region encoding it can be determined, and thereby
the amino acid sequence of the protein of the present invention can
be obtained. Furthermore, genomic DNA can be isolated by screening
a genomic DNA library using the obtained cDNA as the probe.
[0085] Specifically the following method can be followed. First,
mRNA is isolated from cells, tissues, or organs (for example, the
ovary, the testis, the placenta etc.) expressing the protein of the
present invention. The isolation of mRNA can be effected 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 (Chomczynski, P. and Sacchi,
N., Anal. Biochem. (1987) 162, 156-159) and the like, and then by
purifying mRNA from total RNA using the mRNA Purification Kit
(Pharmacia) and the like. mRNA can also be prepared directly by
using the QuickPrep mRNA Purification Kit (Pharmacia).
[0086] cDNA is synthesized from the mRNA obtained 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 5'-Ampli FINDER RACE 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.
[0087] 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, which is then
introduced into E. coli for selection of colonies to prepare the
desired recombinant vector. The base sequence of the desired DNA
may be confirmed by a known method such as the dideoxy nucleotide
chain termination method.
[0088] 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). Furthermore, the DNA of the present
invention may be altered by using commercially available kits or by
known methods. Alternative includes, for example, digestion with a
restriction enzyme, insertion of a synthetic oligonucleotide or a
suitable DNA fragment, addition of a linker, insertion of an
initiation codon (ATG) and/or a stop codon (ATT, TGA or TAG), and
the like.
[0089] The DNA of the present invention includes DNA that
hybridizes to the DNA comprising the base sequence as set forth in
SEQ ID NO: 1 under a stringent condition and DNA that encodes a
protein functionally equivalent to the above protein of the present
invention.
[0090] As the stringent condition, which can be chosen as
appropriate by a person skilled in the art, there can be mentioned
a low stringent condition. A low stringent condition is, for
example, 42.degree. C., 2.times.SSC, and 0.1% SDS, and preferably
50.degree. C., 2.times.SSC, and 0.1% SDS. More preferably, there
can be mentioned a high stringent condition. As a high stringent
condition, for example, there can be mentioned 65.degree. C.,
2.times.SSC, and 0.1% SDS. In these conditions, the higher the
temperature is the higher the homology of DNA obtained is. The
above hybridizing DNA may preferably be naturally occurring DNA,
for example cDNA or chromosomal DNA.
[0091] The present invention also relates to a vector in which the
DNA of the present invention has been inserted. The vector of the
present invention is useful in retaining the DNA of the present
invention in the host cell and in expressing the protein of the
present invention.
[0092] The vector is not specifically limited, as long as it has
florin for use in amplification in E. coli to produce and amplify
in large quantities the vector in E. coli (for example, JM109,
DH5a, HB101, XL1Blue) when E. coli is used as the host, and a
selection gene (for example a drug resistance gene that can be
identified by a drug such as ampicillin, tetracycline, kanamycin,
and chloramphenicol) in the transformed E. coli. Examples of
vectors include M13 vectors, pUC vectors, pBR322, pBluescript,
pCR-Script and the like. Also, for the purpose of subcloning and
excising of DNA, pGEM-T, pDIRECT, pT7 etc., in addition to the
above vector, may be mentioned. When a vector is used to produce
the protein of the present invention specifically, the use of an
expression vector is useful.
[0093] When expression is to be effected in E. coli, the expression
vector should have the above characteristics so that it can be
expressed in E. coli, and besides, when the host is such E. coli as
JM109, DH5a, HB101, XL1-Blue etc., it must have a promoter that
permits efficient expression in E. coli, such as the lacz promoter
(Ward et al., Nature (1989) 341: 544-546; FASEB J. (1992) 6,
2422-2427), the ara B promoter (Better et al., Science (1988) 240,
1041-1043), the T7 promoter, or the like. As such vectors, there
may be mentioned, in addition to the above vectors, pGEX-5X-1
(manufactured by Pharmacia), "QIAexpress system" (manufactured by
Qiagen), pEGFP, .pET (in this case, the host is preferably BL21
that is expressing T7 RNA polymerase), or the like.
[0094] The vector can also contain a signal sequence for
polypeptide secretion. As the signal sequence for protein
secretion, when produced in the periplasm of E. coli, the pe1B
signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379)
can be used. The introduction of a vector into the host cell may be
effected by, for example, the calcium chloride method and the
electroporation method.
[0095] For the production of the protein of the present invention,
in addition to E. coli, there can be mentioned expression vectors
derived from mammals (for example, pcDNA3 (manufactured by
Invitrogen), pEGF-BOS (Nucleic Acids Res. 18 (17), 5322, 1990),
pEF, pCDM8), expression vectors derived from insect cells (for
example, "Bac-to-Bac baculovirus expression system" (manufactured
by GIBCO BRL), pBacPAK8), expression vectors derived from plants
(for example, pMH1, pMH2), expression vectors derived from animal
viruses (for example, pHSV, pMV, pAdexLcw), expression vectors
derived from retrovirus vectors (for example, pZlpneo), expression
vectors derived from yeast (for example, "Pichia Expression Kit"
(manufactured by Invitrogen), pNV11, SP-Q01), expression vectors
derived from Bacillus subtilis (for example, pPL608, pKTH50), and
the like.
[0096] For the purpose of expressing in animal cells such as CHO
cells, COS cells, NIH3T3 cells and the like, it is indispensable
for the vectors to have promoters required for expression in cells
such as SV40 promoter (Mulligan et al., Nature (1979) 277, 108),
MMLV-LTR promoter, EFla promoter (Mizushima et al., Nucleic Acids
Res. (1990) 18, 5322), CMV promoter and the like, and more
preferably to have genes (such as a drug resistant gene that can be
identified by a drug such. as neomycin, G418) for selecting
transformation into the cell. As vectors having such
characteristics, there can be mentioned for example pMAM, pDR2,
pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.
[0097] Furthermore, for the purpose of stably expressing a gene and
amplifying the copy number of a gene in the cell, there may be
mentioned a method in which a vector (for example, pCHOI) having
the DHFR gene is introduced into the CHO cells deficient in the
nucleic acid synthetic pathway to complement the deficiency and is
amplified with methotrexate (MTX). For the purpose of transient
expression of a gene, there may be mentioned a method in which
transformation is effected with a vector (pcD etc.) having the
origin of replication for SV40 using COS cells having on the
chromosome a gene that expresses the SV40 T antigen.
[0098] As the origin of replication, there can be used those
derived from polyoma virus, adenovirus, bovine papilloma virus
(BPV) and the like. Furthermore, for the amplification of gene copy
numbers 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.
[0099] On the other hand, as methods of expressing the DNA of the
present invention in an organism, there can be mentioned a method
in which the DNA of the present invention is integrated into a
suitable vector, and then introduced into an organism by the
retrovirus method, the liposome method, the cationic liposome
method, the adenovirus method and the like. This permits the gene
therapy of diseases associated with mutation in the gene of the
present invention. As the vector used, there can be mentioned, but
not limited to, an adenovirus vector (for example, pAdexlcw), a
retrovirus vector (for example, pZlpneo) and the like. Common
genetic manipulation such as the insertion of the DNA of the
present invention into a vector may be performed by a standard
method (Molecular Cloning, 5.61-5.63). The administration into an
organism may be an ex vivo method or an in vivo method.
[0100] The present invention also relates to a host cell into which
the vector of the present invention has been introduced. The host
cell into which the vector of the present invention is introduced
is not specifically limited, and E. coli and various animal cells
may be used. For example, the host cell of the present invention
can be used for the production of the protein of the present
invention or as a production system for expression. The production
system of protein preparation comprises the in vitro and the in
vivo production system. As an in vitro production system, there can
be mentioned a production system which employs eukaryotic cells and
a production system which employs prokaryotic cells.
[0101] 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 mammalian cells such as
CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells,
myeloma cells, baby hamster kidney (BHK) cells, HeLa cells and Vero
cells, amphibian cells such as Xenopus oocytes (Valle, et al.,
Nature (1981) 291, 358-340), or insect cells such as sf9, sf21, and
Tn5.
[0102] 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-i (Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275)
can be preferably used. In animal cells, for the purpose of large
scale production, CHO cells are specifically preferred. The
introduction of a vector into the host cell may be carried out by,
for example, the calcium phosphate method, the DEAE-dextran method,
the cationic ribozome DOTAP (manufactured by Boehringer Mannheim),
the electroporation method, and the lipofection method.
[0103] As plant cells, for example, there are known cells derived
from Nicotiana tabacum as the protein production system, which may
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 niger.
[0104] When the prokaryotic cells are used, there are the
production systems which employ bacterial cells. Known bacterial
cells include Escherichia coli (E. coli) such as JM109, DH5a and
HB101, and Bacillus subtilis.
[0105] By transforming these cells with the desired DNA and
culturing the transformed cells in vitro, proteins can be obtained.
Culturing is conducted using known methods. For example, as the
culture liquid, DMEM, MEM, RPMI1640, and IMDM can be used, and
hereupon 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 about 30 to 40.degree. C. for about 15 to 200 hours with, as
desired, medium change, aeration, and agitation.
[0106] On the other hand, as in vivo production systems, there can
be mentioned those which employ animals and those which employ
plants. The DNA of interest is introduced into these animals or
plants, and the proteins are produced in the body of such animals
or plants, and recovered. The term "host cell" as used herein
encompasses these animals and plants.
[0107] When animals are used, there are the production systems
which employ mammals and insects. As mammals, goats, pigs, sheep,
mice, and cattle can be used (Vicki Glaser, SPECTRUM Biotechnology
Applications, 1993). When mammals are used, transgenic animals can
be used.
[0108] For example, the DNA of interest is prepared as a fusion
gene with a gene encoding protein which is inherently produced in
the milk such as goat casein. The DNA fragment containing the
fusion gene into which the DNA has been inserted is injected into a
goat embryo, and the embryo is introduced into a female goat. The
protein of interest can be obtained from the milk produced by the
transgenic goat borne to the goat who received the embryo or the
offspring thereof. In order to increase the amount of milk
containing the protein 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).
[0109] 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 protein can be
obtained from the body fluid of the silkworm (Susumu, M. et al.,
Nature (1985) 315, 592-594).
[0110] 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 tobacco such as
Nicotiana tabacum to obtain the desired protein from the leaves of
the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24,
131-138)
[0111] Proteins of the present invention thus obtained can be
separated from the inside or outside (culture medium etc.) of the
host cell and then may be purified as virtually pure and
homogeneous proteins. Separation and purification of the antibody
for use in the present invention may be accomplished by, but not
limited to, separation and the purification methods conventionally
used for protein purification. Proteins can be separated and
purified by selecting and combining, as appropriate, methods
including chromatography columns, filtration, ultrafiltration,
salting-out, solvent precipitation, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric focusing, dialysis, recrystallization and the like.
[0112] 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,
1996). These chromatographies can be carried out using a liquid
chromatography such as HPLC and FPLC. The present invention
encompasses proteins that were highly purified using these
purification methods.
[0113] Furthermore, before or after purification, proteins can be
modified or peptides can be partially removed as appropriate by
acting a suitable protein modifying enzyme. As the protein
modifying enzyme, there can be used, for example, trypsin,
chymotrypsin, lysyl endopeptidase, protein kinase, and
glucosidase.
[0114] The present invention also relates to antibody that binds to
the protein of the present invention. The form of the protein of
the present invention is not specifically limited, and includes
polyclonal antibody as well as monoclonal antibody. Furthermore,
antiserum obtained by immunizing an immune animal such as a rabbit
with the protein of the present invention, all classes of
polyclonal antibodies and monoclonal antibodies, and moreover human
antibodies and humanized antibodies by gene recombinant
technology.
[0115] Though the protein of the present invention for use as 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 mammals such as humans, mice, or rats with
the human-derived protein being most preferred. Human-derived
protein can be obtained using the gene sequence or the amino acid
sequence disclosed herein.
[0116] Proteins that are used as the sensitizing antigen herein may
be complete proteins or partial peptides thereof. As the partial
peptides of the proteins, for example, N-terminal fragments (N) or
C-terminal fragments (C) of the proteins may be mentioned. The term
"antibody" as used herein means antibody that reacts to the
full-length protein or fragments thereof.
[0117] A gene encoding the protein of the present invention or a
fragment thereof may be inserted into a known expression system,
and said vector is used to transform the host cell described herein
to obtain the protein of interest or a fragment thereof from the
inside or outside of said host cell by a known method, which may be
used as the sensitizing antigen. Alternatively, the cell that
expresses the protein or lysates thereof or the chemically
synthesized protein of the present invention may be used as
sensitizing antigen.
[0118] 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 animals of the order
Rodentia, the order Lagomorpha, and the order Primates. Animals of
order Rodentia include, for example, mice, rats, hamsters, and the
like. Animals of order Lagomorpha include, for example, rabbits.
Animals of order 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.
[0119] Immunization of animals with a sensitizing antigen is
carried out using a known method. A general method, for example,
involves the intraperitoneal or subcutaneous injection 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
is mixed, as desired, with an appropriate amount of a common
adjuvant, for example Freund's complete adjuvant. After being
emulsified, it is administered to the mammal. Then the sensitizing
antigen dissolved at a suitable amount is Freund's incomplete
adjuvant is administered 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.
[0120] 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 by a known method. As
polyclonal antibodies, serum containing the polyclonal antibodies
may be used, or, as desired, the fraction containing the polyclonal
antibodies may be isolated from the serum and used. For example,
using an affinity column to which the protein of the present
invention has been coupled, a fraction that recognizes the protein
of the present invention only is obtained, and this fragment is
purified using a Protein A or a Protein G column, to prepare an
immunoglobulin G or M.
[0121] In order to obtain monoclonal antibodies, immune cells of
the mammal that was sensitized with the above 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 cells. The mammalian myeloma cells as the
other parent cells which are subjected to cell fusion with the
above-mentioned immune cells preferably include myeloma cells of a
mammal, more preferably myeloma cells that have acquired a
characteristic feature for the selection of fusion cells by a
drug.
[0122] Cell fusion between the above immune cells and the myeloma
cells may be carried out according to the conventional method, for
example, the method of Milstein (Galfre, G. and Milstein. C,
Methods Enzymol. (1981) 73, 3-46) and the like.
[0123] The hybridoma obtained by cell fusion may be selected by
culturing in a 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 for 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.
[0124] 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, for example lymphocytes infected
with EB virus, with a protein, 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 that produces the desired human antibody having the
activity of binding the protein (see Japanese Unexamined Patent
Publication (Kokai) No. 63-17688).
[0125] Then, the hybridoma obtained is transplanted into the
abdominal cavity of a mouse, ascites is recovered from the mouse,
and the monoclonal antibody obtained is purified and prepared by
subjecting it to ammonium sulfate precipitation, Protein A or
Protein G column, DEAE ion exchange chromatography, affinity column
to which the protein of the present invention has been coupled, and
the like. The antibody of the present invention can be used for the
purification and detection of the protein of the present invention
and, besides, becomes a candidate for the agonist and the
antagonist of the protein of the present invention. The antibody
may be used for antibody therapy of diseases with which the protein
of the present invention is associated. When the antibody obtained
is used for the purpose (antibody therapy) of administering to a
human body, it is preferably human antibody or humanized antibody
in order to reduce immunogenicity.
[0126] Furthermore, a transgenic animal having a repertoire of
human antibody genes is immunized with the antigen protein, cells
expressing them or lysates thereof to obtain the antibody-producing
cells, which are used to obtain human antibody against the protein
for use in the present invention using hybridomas 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).
[0127] In addition to using a hybridoma to produce antibody,
antibody-producing immune cells such as sensitized lymphocytes that
have been immortalized with an oncogene may be used to obtain
antibody.
[0128] A monoclonal antibody thus produced can also be obtained as
a recombinant antibody by recombinant gene technology (see, for
example, Borrebaeck, C. A. K., and Larrick, J. W., THERAPEUTIC
MONOCLONAL ANTIBODIES, published in the United Kingdom by MACMILLAN
PUBLISHERS LTD., 1990). Recombinant antibody may be produced by
cloning DNA encoding it from the hybridoma or an immune cell such
as sensitized lymphocytes that produce antibodies, and integrating
it into a suitable vector, which is then introduced into a host to
produce said antibody. The present invention also encompasses such
recombinant antibodies.
[0129] Antibodies of the present invention may be antibody
fragments or modified versions thereof as long as they bind the
protein of the present invention. For example, as fragments of
antibody, there may be mentioned Fab, F(ab')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 (Huston, J. S. et al., Proc. Natl.
Acad. Sci. USA (1988) 85, 5879-5883). 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
then 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; Pluckthun, 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).
[0130] As modified antibodies, antibodies bound to various
molecules such as polyethylene glycol (PEG) can also be used. The
term "antibody" as used herein encompasses these modified
antibodies. In order to obtain these modified antibodies, the
antibody obtained may be chemically modified. These methods are
established in the field of the art.
[0131] Furthermore, the antibody of the present invention may be
obtained as a chimeric antibody comprising a variable region
derived from a non-human antibody and a constant region derived
from a human antibody, or as a humanized antibody comprising a
complementarity determining region (CDR) derived from a non-human
antibody and a framework region (FR) and a constant region derived
from a human antibody.
[0132] Antibodies obtained as described above can be purified to
homogeneity. Separation and purification of the antibody for use in
the present invention may be accomplished by the separation and the
purification methods conventionally used for protein. For example,
antibody can be separated and purified by selecting and combining,
but not limited to, chromatography columns such as affinity
chromatography, filtration, ultrafiltration, salting-out, dialysis,
SDS polyacrylamide gel electrophoresis, and isoelectric focusing as
appropriate (Antibodies: A Laboratory Manual, Ed Harlow and David
Lane, Cold Spring Harbor Laboratory, 1988). The measurement of
concentration of the antibody obtained as above can be determined
by the measurement of absorbance, enzyme-linked immunosorbent assay
(ELISA), and the like.
[0133] As columns for use in affinity chromatography, there can be
mentioned a Protein A column and a Protein G column. Examples of
the columns used in the Protein A column are Hyper D, POROS,
Sepharose F. F. (Pharmacia) and the like.
[0134] 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, 1996). These chromatographies can be
carried out using a liquid chromatography such as HPLC and
FPLC.
[0135] As the method of determining antibody binding activity of
the antibody of the present invention obtained as above, there can
be used enzyme-linked immunosorbent assay (ELISA),
enzymeimmunoassay (EIA), radioimmunoassay (RIA), or fluorescent
antibody assay. When ELISA is used, the protein of the present
invention is added to a plate immobilized with the antibody of the
present invention, and then a sample containing the antibody of
interest, for example a culture supernatant of the
antibody-producing cells or purified antibody is added.
[0136] The antigen binding activity can be evaluated by adding a
second antibody that recognizes the antibody labeled with an enzyme
such as alkaline phosphatase, incubating and then washing the
plate, and subsequently, after adding the enzyme substrate such as
p-nitrophenyl phosphate thereto, determining absorbance. As the
protein, a fragment of the protein, a fragment comprising the
C-terminal thereof, or a fragment comprising the N-terminal thereof
may be used. For the evaluation of activity of the antibody of the
present invention, BIAcore (Pharmacia) can be used.
[0137] By using these techniques, the method of detecting or
determining the protein of the present invention can be performed,
said method comprising bringing the antibody of the present
invention into contact with a sample expected to contain the
protein of the present invention, and detecting or determining an
immune complex of said antibody and said protein or immunostaining
the cells that express said protein using said antibody.
[0138] The method of detecting or determining the protein of the
present invention can specifically detect or determine the protein
and, therefore, is useful in various experiments using the
protein.
[0139] The present invention also relates to DNA that specifically
hybridizes to DNA (SEQ ID NO: 1) encoding the human WT1 interacting
protein or DNA complementary to said DNA, and that has a chain
length of at least 15 bases. The term "specifically hybridize"
means that there occurs no significant cross-hybridization with DNA
encoding other proteins under a standard hybridization conditions,
preferably under a stringent hybridization condition. Such DNA
includes a probe, a primer, a nucleotide or a nucleotide derivative
(for example, antisense oligonucleotide or ribozyme) that can
specifically hybridizes to DNA encoding the protein of the present
invention or DNA complementary to said DNA. Furthermore, such DNA
can be used for the preparation of DNA chips.
[0140] The present invention includes, for example, an antisense
oligonucleotide that hybridizes to any site in the base sequence of
SEQ ID NO: 1. The antisense oligonucleotide is preferably an
antisense oligonucleotide against at least 15 or more contiguous
nucleotides in the base sequence as set forth in SEQ ID NO: 1. More
preferably, it is the above antisense oligonucleotide in which the
above contiguous at least 15 or more nucleotides contain a
translation initiation codon.
[0141] As the antisense oligonucleotide, their derivatives or
modified versions may be used. As such modified versions, there can
be mentioned, for example, lower alkylphosphonate modified versions
such as methyl phosphonate or ethyl phosphonate type,
phosphorothioate modified versions, or phosphoramidate modified
versions.
[0142] As used herein "antisense oligonucleotide" may contain one
or a plurality of nucleotide mismatches as long as nucleotides
corresponding to nucleotides constituting a given region of DNA or
mRNA are all complementary, and DNA or mRNA and the oligonucleotide
can specifically hybridize to the base sequence as set forth in SEQ
ID NO: 1.
[0143] Such DNA is a region of at least 15 contiguous nucleotide
sequence and has a homology of at least 70%, at least 80%, and at
least 90%, and more preferably 95% or more on the base sequence.
The algorithm used for determining homology may be one described
herein. Such DNA is useful as a probe for detecting or isolating
DNA encoding the protein of the present invention and as a primer
for amplifying it as described below.
[0144] The antisense oligonucleotide derivative of the present
invention binds to DNA or mRNA encoding said protein by acting on
the cells that produce the protein of the present invention,
thereby inhibit its transcription or translation, or promote the
decomposition of mRNA, resulting in the suppression of expression
of the protein of the present invention. Eventually it exhibits an
effect of suppressing the actions of the protein of the present
invention.
[0145] The antisense oligonucleotide derivative of the present
invention can be mixed with an appropriate base that is inert
thereto to formulate an external preparation such as a liniment, a
cataplasm and the like.
[0146] It can also be mixed, as desired, with an excipient, an
isotonic agent, a solubilizer, a stabilizer, an antiseptic, a
soothing agent or the like to formulate a tablet, powders,
granules, a capsule, a liposome capsule, an injection, a solution,
a nasal drop, and the like as well as a lyophilized preparation.
They can be prepared according to conventional methods.
[0147] The antisense oligonucleotide derivative of the present
invention may be applied to the patient by either directly
administering to the affected area of the patient or administering
into the blood vessel thereby allowing the substance to be
delivered to the affected area. Furthermore, an antisense
encapsulating material that enhances prolonged action and membrane
permeability may be used. There may be mentioned, for example,
liposome, poly-L-lysine, lipid, cholesterol, lipofectin or
derivatives thereof.
[0148] Preferably the dosage of the antisense oligonucleotide
derivative of the present invention can be adjusted and a preferred
amount can be employed as appropriate depending on the condition of
the patient. For example, a preferred dosage is in the range of 0.1
to 100 mg/kg, preferably 0.1 to 50 mg/kg.
[0149] The antisense oligonucleotide of the present invention is
useful in inhibiting the expression of the protein of the present
invention, and thereby in suppressing the biological activity of
the protein of the present invention. An inhibitor of expression
containing the antisense oligonucleotide of the present invention
can suppress the biological activity of the protein of the present
invention, and therefore, is useful.
[0150] The protein of the present invention is useful for screening
compounds that bind thereto. Thus, it is used in a method of
screening a compound that binds to the protein of the present
invention, said method comprising bringing the protein of the
present invention into contact with a test sample expected to
contain a compound that binds to said protein, and selecting a
compound having an activity of binding to the protein of the
present invention.
[0151] The protein of the present invention for use in the
screening may be a recombinant protein or a naturally occurring
protein. Alternatively it may be a partial peptide. Test samples
include, but are not limited to, cell extracts, cell culture
supernatants, microbial fermentation products, marine organism
extracts, plant extracts, purified or roughly purified proteins,
peptides, non-peptide compounds, synthetic compounds, and naturally
occurring compounds. The protein of the present invention to be
contacted with the test sample can be contacted with the test
sample as a purified protein, as a form bound to a carrier, and as
a fusion protein with another protein.
[0152] As a method of screening a protein (ligand etc.) that binds
to the present invention using the protein of the present
invention, various methods known to a person skilled in the art can
be used. Such screening can be performed by, for example,
immunoprecipitation. Specifically, it can be carried out as
follows. A gene encoding the protein of the present invention is
inserted into a vector for expressing foreign genes such as
pSV2neo, pcDNA I and pCD8 so as to permit the expression of said
gene in an animal cell.
[0153] As the promoter used in expression, any commonly used
promoters may be used such as:SV40 early promoter (Rigby In
Williamson (ed.), Genetic Engineering, Vol. 3. Academic Press,
London, pp. 83-141 (1982), EF-1a promoter (Kim et al., Gene 91,
217-223, 1990), CAG promoter (Niwa et al., Gene 108, 193-200,
1991), RSV LTR promoter (Cullen Methods in Enzymology 152, 684-704,
1987), SR a promoter (Takebe et al., Mol. Cell. Biol. 8, 466,
1988), CMV immediate early promoter (Seed and Aruffo, Proc. Natl.
Acad. Sci. USA 84, 3365-3369, 1987), SV40 late promoter (Gheysen
and Fiers, J. Mol. Appl. Genet. 1, 385-394, 1982), Adenovirus late
promoter (Kaufman et al., Mol. Cell. Biol. 9, 946, 1989), and HSV
TK promoter.
[0154] In order to express a foreign gene by introducing the gene
into an animal cell, there are the electroporation method (Chu, G.
et al., Nucleic Acids Res. 15, 1311-1326, 1987), the calcium
phosphate method (Chen, C and Okayama, H., Mol. Cell. Biol. 7,
2745-2752, 1987), the DEAE dextran method (Lopata, M. A. et al.,
Nucleic Acids Res. 112, 5707-5717, 1984; Sussman, D. J. and Milman,
G., Mol. Cell. Biol. 4, 1642-1643, 1985), the lipofectin method
(Derijard, B., Cell 7, 1025-1037, 1994; Lamb, B. T. et al., Nature
Genetics 5, 22-30, 1993; Rabindran, S. K. et al., Science 259,
230-234, 1993), and the like, and any of them may be used.
[0155] By introducing a recognition site (epitope) of a monoclonal
antibody, of which the specificity is known, into the N-terminal or
C-terminal of the protein of the present invention, the protein of
the present invention can be expressed as a fusion protein having
the recognition site of the monoclonal antibody. As the
epitope-antibody system, commercially available ones may be used
(Jikken Igaku (Experimental Medicine), 13, 85-90, 1995). Vectors
are commercially available that permit the expression of fusion
proteins with p-galactosidase, maltose-binding protein, glutathione
S-transferase, green fuorescence protein (GFP), and the like, via
multiple cloning sites.
[0156] A method of preparing a fusion protein has also been
reported in which a small epitope portion alone comprising a few to
about a dozen amino acids is introduced so that the effect of the
resulting fusion protein on changes in the property of the protein
of the present invention is minimal. For example, an epitope such
as poly-histidine (His-tag), influenza hemagglutinin (HA), human
c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7
genelo protein (T7-tag), herpes simplex virus glycoprotein
(HSV-tag), E-tag (epitope on the monoclonal phage) and the like and
the monoclonal antibody that recognizes it can be used as the
epitope-antibody system for screening proteins that bind to the
protein of the present invention (Jikken Igaku (Experimental
Medicine) 13, 85-90, 1995).
[0157] In immunoprecipitation, an immune complex is formed by
adding these antibodies to a cell lysate prepared by using a
suitable surfactant. The immune complex comprises the protein of
the present invention, a protein capable of binding thereto, and an
antibody. In addition to using an antibody to the above epitope, an
antibody to the protein of the present invention can also be used
to perform immunoprecipitation. The antibody to the protein of the
present invention can also be prepared by introducing a gene
encoding the protein of the present invention into a suitable E.
coli expression vector to express said protein in E. coli,
purifying the expressed protein, and then using this to immunize a
rabbit, a mouse, a rat, a goat, a chicken, and the like. It can
also be prepared by immunizing the above animal with a partial
peptide of the protein of the present invention that was
synthesized. An immune complex can be precipitated by using, for
example, Protein A Sepharose or Protein G Sepharose when the
antibody is a mouse IgG antibody. When the protein of the present
invention was prepared as a fusion protein with an epitope such as
GST, an immune complex can be formed, as when the antibody of the
protein of the present invention was used, by using a substance
that specifically binds to these epitopes such as
glutathione-Sepharose 4B.
[0158] For a general method of immunoprecipitation, it is carried
out according to, or pursuant to, the method described in an
article (Harlow, E. and Lane, D.: Antibodies, pp. 511-552, Cold
Spring Harbor Laboratory publications, New York, 1988).
[0159] For the analysis of immunoprecipitated protein, SDS-PAGE is
generally used, in which the bound protein can be analyzed based on
the molecular weight of the protein by using a suitable
concentration of gel. At this time, generally the protein bound to
the protein of the present invention cannot be detected by a
standard staining method for protein such as Coomassie staining or
silver staining, and therefore the cells are cultured in a culture
liquid containing .sup.35S-methionine or .sup.35S-cycteine to label
the protein in the cell, which is then detected in order to enhance
the sensitivity of detection. If the molecular weight of the
protein is known, the protein of interest can be directly purified
from the SDS-polyacrylamide gel electrophoresis, and the sequence
can be determined.
[0160] As a method of isolating a protein bound to said protein
using the protein of the present invention, the west western blot
(Skolnik, E. Y. et al., Cell (1991) 65, 83-90), for example, can be
used. Thus, a cDNA library is constructed using a phage vector
(.lamda.gt11, ZAP etc.) from a cell, a tissue, and an organ (for
example, tissues, cells, and cultured cells such as the heart, the
placenta, the testis, the thymus, and peripheral leukocytes) in
which the binding protein that binds to the protein of the present
invention is expected to be expressed, and to the library is
expressed on the LB-agarose to immobilize the expressed protein on
a filter. Ten the purified and labelled protein of the present
invention and the above filter are reacted, and a plaque that
expresses the protein bound to the protein of the present invention
is detected based on the label.
[0161] As methods of labelling the protein of the present
invention, there can be mentioned a method of utilizing the binding
property of biotin and avidin, a method of utilizing an antibody
that specifically binds to the protein of the present invention or
a peptide or a polypeptide (for example, GST) fused to the protein
of the present invention, a method of utilizing radioisotope, a
method of utilizing fluorescence, and the like.
[0162] As another aspect of the screening method of the present
invention, there can be mentioned a method of conducting a 2-hybrid
system (Fields, S. and Sternglanz, R., Trends. Genet. (1994) 10,
286-292) that uses cells. The protein of the present invention is
fused to the SRF DNA binding region or the GAL4 DNA binding region,
and then is expressed in yeast cells. From the cells expected to
express the protein that binds to the protein of the present
invention, a cDNA library is constructed that expresses, in a form
fused with the VP16 or the GAL4 transcription activation region,
which is introduced into the above yeast cells. From the positive
clones detected, library-derived cDNA is isolated and introduced
into E. coli for expression (when the protein that binds to the
protein of the present invention is expressed in the yeast cells,
the binding of the two results in the activation of the reporter
gene, and thereby positive clones can be confirmed.).
[0163] It is also possible to prepare a protein that binds to the
protein of the present invention or the gene thereof using the
"two-hybrid system" (MATCHMARKER Two-Hybrid System", "Mammalian
MATCHMARKER Two-Hybrid Assay Kit", "MATCHMARKER One-Hybrid System"
(all manufactured by Clontech), "HybriZAP Two-Hybrid Vector System"
(manufactured by Stratagene), and an article "Dalton S. and
Treisman R. (1992) Characterization of SAP-1, a protein recruited
by serum response factor to the c-fos serum response element. Cell
68, 597-612"). As the reporter gene, the Ade2 gene, the LacZ gene,
the CAT gene, the luciferase gene, the PAI-1 (plasminogen activator
inhibitor type 1) gene, and the like can be used in addition to the
H1S3 gene.
[0164] The screening of a compound that binds to the protein of the
present invention can also be effected using affinity
chromatography. For example, the protein of the present invention
is immobilized onto an affinity column, to which a test sample
expected to be expressing a protein that binds to the protein of
the present invention is applied. As the test sample in this case,
for example, cell extracts, cell lysates and the like may be
mentioned. After applying the test sample, the column is washed,
and the protein that is bound to the protein of the present
invention can be prepared.
[0165] The protein obtained is analyzed for its amino acid sequence
and, based on the sequence, an oligo DNA is synthesized. Using said
DNA as the probe, a DNA library can be screened so that DNA
encoding said protein can be obtained.
[0166] In accordance with the present invention, as a means to
detect or determine the compound that is bound, a biosensor that
utilizes the surface plasmon phenomenon can be used. The biosensor
that utilizes the surface plasmon phenomenon permits realtime
observation, as a surface plasmon resonance signal, of the
interaction between the protein of the present invention and the
test compound using a trace amount of protein without labeling it
(for example, BIAcore, manufactured by Pharmacia). Thus, by using a
biosensor such as BIAcore, the binding of the protein of the
present invention and the test compound can be evaluated.
[0167] As methods of isolating compounds that bind to the protein
(including an agonist and an antagonist) of the present invention,
in addition to protein, a method in which a synthetic compound, a
natural product bank, or a random phage peptide display library is
acted onto the immobilized protein of the present invention, and
molecules that bind to the protein of the present invention are
screened, and a screening method using a high throughput using the
combinatorial chemistry technology (Wrighton N C; Farrell F X;
Chang R; Kashyap A K; Barbone F P; Mulcahy L S; Johnson D L; Barret
R W; Jolliffe L K; Dower W J., Small peptides as potent mimetics of
the protein hormone erythropoietin, Science (United States) Jul.
26, 1996, 273 pp. 458-64, Verdine G L., The combinatorial chemistry
of nature, Nature (England) Nov. 7, 1996, 384 pp. 11-13, Hogan J C
Jr., Directed combinatorial chemistry, Nature (England) Nov. 7,
1996, 384 pp. 17-9) are known to a person skilled in the art.
[0168] Furthermore, the present invention relates to a method of
screening a compound that promotes or inhibits the activity of
protein of the present invention. As the WT1 interacting protein of
the present invention has an activity of binding to the WT1 protein
and thereby regulating the function of the WT1 protein, this
activity can be used as an index to screen a compound that promotes
or inhibits the activity of the WT1 interacting protein of the
present invention.
[0169] The screening method comprises the steps of (a) culturing
the cells that express the WT1 interacting protein in the presence
of a sample to be tested, (b) detecting the growth of said cells,
and (c) selecting a compound that promotes or inhibits said growth
as compared to when detected in the absence of a sample to be
tested.
[0170] The protein used in screening is not specifically limited as
long as it has an activity of regulating the function of the WT1
protein. For example, the human WT1 interacting protein can be
mentioned, and a protein that is functionally equivalent to this
protein can also so be used. Also, the WT1 interacting protein may
be intracellularly or extracellularly expressed by the cell.
[0171] Test samples are not specifically limited, and there may be
mentioned cell extracts, cell culture supernatants, microbial
fermentation products, marine organism extracts, plant extracts,
purified or roughly purified proteins, peptides, non-peptide
compounds, synthetic compounds, and naturally occurring compounds.
It is also possible to use the compound obtained in the screening
of compounds that bind to the protein of the present invention, as
the test compound.
[0172] The compound isolated in this screening can be an agonist or
an antagonist candidate for the protein of the present invention.
The term "agonist" as used herein means a molecule that activates
the function of the protein of the present invention by binding
thereto. Also the term "antagonist" as used herein means a molecule
that suppresses the function of the protein of the present
invention by specifically binding thereto. Furthermore, it can be a
candidate compound that inhibits the interaction of the molecule
(including DNA and protein) that interacts with the protein of the
present invention.
[0173] The detection of cell growth can be effected by, for
example, detecting the measurement of colony forming rate and the
determination of the growth rate of the cell, the measurement of
cell cycle, and the like.
[0174] The compounds isolated from these screenings can become
candidate drugs for promoting or inhibiting the activity of the
protein of the present invention, and its application into the
treatment of diseases (for example cancer) with which the protein
of the present invention is associated, is conceivable.
[0175] Substances, obtained using the screening method of the
present invention, in which part of the structure of compounds that
promote or inhibit the activity of the WT1 interacting protein has
been modified by addition, deletion and/or substitution is also
included in the compounds obtained by the screening method of the
present invention.
[0176] The present invention also relates to a method of screening
a compound that promotes or inhibits the binding of a WT1
interacting protein of the present invention with the WT1 protein.
The method comprises the steps of: (a) allowing the WT1 interacting
protein to react with the WT1 protein in the presence of a sample
to be tested, (b) determining the binding activity of both
proteins, and (c) selecting a compound that promotes or inhibits
said binding as compared to when detected in the absence of the
sample to be tested.
[0177] As a method of assaying whether a compound contained in the
test sample promotes or inhibits the binding of the present protein
and the WT1 protein, there may be mentioned the west western
blotting method or a method of quantitating the conjugate of the
both proteins in a solution (in vitro) or in the cell (in
vivo).
[0178] When the compound obtained by the screening method of the
present invention is used as a drug for humans and mammals such as
mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep,
pigs, cattle, monkeys, baboons, and chimpanzees, the isolated
compound can also be formulated according to a known pharmaceutical
method for administration, in addition to directly administering
the compound itself to patients.
[0179] For example, it can be used either orally as sugar-coated
tablets, capsules, elixirs or microcapsules, or parenterally in the
form of aseptic solutions with water or another pharmaceutically
acceptable liquid or injections of a suspension. For example,
conceivably, it is combined with a pharmaceutically acceptable
carrier or a medium, specifically sterile water or physiological
saline, a plant oil, an emulsifying agent, a suspension, a
surfactant, a stabilizer, a flavoring agent, an excipient, a
vehicle, a preservative, a binder and the like, and mixed in a unit
dosage form, required for commonly recognized pharmaceutical
practice, to formulate into a drug. The amount of an active
ingredient in these formulations is adjusted to provide a suitable
amount in the indicated range.
[0180] As additives that can be mixed into tablets or capsules,
there can be used, for example, a binder such as gelatin, corn
starch, gum tragacanth and gum Arabic, an excipient such as
crystalline cellulose, a swelling agent such as corn starch,
gelatin and alginic acid, a lubricant such as magnesium stearate, a
sweetening agent such as sucrose, lactose and saccharin, and a
flavoring agent such as peppermint, gaultheria oil or cherry. When
the formulation dosage unit is a capsule, the above materials can
further contain liquid carriers like oil and fat. Sterile
compositions for injection can be formulated according to a normal
pharmaceutical practice using a vehicle such as distilled water for
injection.
[0181] As an aqueous solution for injection, there can be
mentioned, for example, physiological saline, an isotonic liquid
containing glucose and another adjuvant, for example, D-sorbitol,
D-mannose, D-mannitol and sodium chloride, and may be combined with
a suitable solubilizing agent such as alcohol, specifically
ethanol, polyalcohol such as propylene glycol, polyethylene glycol,
non-ionic surfactant such as polysorbate 80 (TM), and HCO-50.
[0182] As an oleaginous solution, there may be mentioned sesame oil
and soy bean oil, and as a solubilizing solution, benzyl benzoate
or benzyl alcohol may be used in combination. Furthermore, a buffer
such as phosphate buffer, sodium acetate buffer, a soothing agent
such as procaine chloride, a stabilizer such as benzyl alcohol and
phenol, an antioxidant may be blended. The prepared injection is
usually filled into a suitable ampoule.
[0183] The administration to patients may be performed by
intraarterial injection, intravenous injection, subcutaneous
injection, and nasally, transbronchially, intramuscularly, or
orally by a method known to a person skilled in the art. The dosage
may vary depending on the weight and age of patients, method of
administration and the like, and a person skilled in the art can
select a suitable dosage as appropriate. Gene therapy is also
contemplated in which said DNA is integrated into a vector for gene
therapy. The dosage method of administration may vary depending on
the weight and age of patients, and the like, and a person skilled
in the art can select a suitable dosage as appropriate.
[0184] For example, the dosage of a compound that binds to the
protein of the present invention or a compound that inhibits the
activity of the protein of the present invention may vary depending
on the disease condition, but in the case of oral administration,
dosage for an adult (assuming body weight being 60 kg) is about 0.1
to 100 mg/day, preferably about 1.0 to 50 mg, and more preferably
about 1.0 to 20 mg.
[0185] In the case of the parenteral administration, the unit
dosage may vary depending on the subject to be administered, the
subject organ, disease condition, and the method of administration,
but in the form of an injection, a convenient dosage for an adult
(assuming body weight being 60 kg) is about 0.01 to 30 mg/day,
preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10
mg injected intravenously. For other animals, the amount converted
from body weight of 60 kg may be administered.
EXAMPLES
[0186] Now the present invention will be explained in more detail
hereinbelow.
Example 1
Purification of WTIP
[0187] (1) Construction and Purification of GST Fusion Protein
[0188] Using pBluescript II/WT1 (+/+) (Kudoh T. et al., Oncogene,
Vol. 13, pp. 1431-1439, 1996) as the template, PCR was conducted
using the following primers: TABLE-US-00001 1) GST-WT2
5'-TTGAATTCAATGGGCTCCGACGTGCGG-3' (SEQ ID NO:3)
5'-TTGTCGACCATGGGATCCTCATGCTT-3' (SEQ ID NO:4) 2) GST-WT3
5'-TTGAATTCAATGGGCTCCGACGTGCGG-3' (SEQ ID NO:5)
5'-TTGTCGACGAAGACACCGTGCGTGTG-3' (SEQ ID NO:6) 3) GST-WT4
5'-TTGAATTCAGATCCAATGGGCCAGCAC-3' (SEQ ID NO:7)
5'-TTGTCGACGAAGACACCGTGCGTGTG-3' (SEQ ID NO:8)
[0189] in which an EcoRI recognition sequence has been added to the
5'-end and a NotI recognition sequence has been added to the 3'-end
to construct insertion fragments. To the regions that are difficult
to amplify, restriction enzyme fragments were inserted via a
plasmid as appropriate. The base sequence of the amplified fragment
is 1-882 for GST-WT3, 1-546 for GST-WT2, and 541-882 for GST-WT4 (B
in FIG. 1). The insertion fragment was integrated into pGEX-5X-3
(Amersham Pharmacia Biotech) and then the sequence was confirmed,
and transformed into an E. coli strain BL21 (DE3). At this time,
pT-Trx, a thioredoxin expression vector, was coexpressed in order
to enhance the solubility of the fusion protein (Yasukawa T. et
al., J. Biol. Chem. Vol. 270, pp. 25328-25331, 1995).
[0190] After confirming expression, an overnight culture was
diluted 10-fold and incubated at 37.degree. C. for 1.5 hour, to
which isopropyl-(3-D-thiogalacto-pyranoside (IPTG) was added to a
final concentration of 0.1 mM. After further culturing for 5 hours,
the cells were collected, to which a cell dissolution buffer (50 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM Pefabloc S C
(Boehringer Mannheim), 10 .mu.g/ml leupeptin, 1 mM DTT) was added,
and solubilized by sonication. The fusion protein in the
supernatant was bound to the glutathione Sepharose 4B (Amersham
Pharmacia Biotech), and eluted with an elution buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM reduced glutathione, 1 mM
DTT). The protein concentration was determined by the Bradford
method (Protein Assay, Bio-Rad) and bovine serum albumin was used
as a standard.
[0191] (2) Detection of WTIP by West Western Blot
[0192] In order to investigate the presence of the WT1 interacting
protein that can be detected with the GST fusion protein in the
K562 cells, K562 (5.times.10.sup.6) was dissolved in 500 .mu.l of
the sample buffer for SDS-PAGE, and the west western blot was
carried out.
[0193] After separating the sample protein on SDS-PAGE, it was
transferred to a PVDF membrane (Immobilon-P, Millipore) by the
semi-dry method, blocked with TBST (0.05% Tween 20) containing 2%
skim milk, and was reacted with 10-20 p.g/ml TBS for 1 hour to
overnight.
[0194] After washing the PVDF membrane with TEST, it was reacted
for 1 hour with a 1000-fold dilution of anti-GST antibody (Santa
Cruz) in TBST, washed again in TEST, and then reacted for 1 hour
with a 8000-fold dilution of anti-mouse IgG antibody (alkaline
phosphatase (ALP)-conjugated, or horseradish peroxidase
(HRP)-conjugated) in TEST. After washing in TBST and TBS, it was
subjected to color development with a NBT/BCIP solution for the
ALP-conjugated antibody, and was subjected to light emission with
ECL for the HRP-conjugated antibody.
[0195] FIG. 2 shows the result of the west western blot. A band at
about 115 kDa observed in GST-WT2 and GST-WT3 was not recognized in
GST, indicating that this is a specific band showing the presence
of the WT1 interacting protein. Furthermore, since the same band
was not recognized in GST-WT4, it was shown to have a binding site
in the 182 amino acid residues at the N-terminal end of WT1. Since
there had been no reports on the WT1 interacting protein of this
size (p 115), its separation and purification were attempted.
[0196] (3) Purification of WTIP
[0197] Sample preparation
[0198] After washing the K562 cells (6.times.10.sup.8 cells) (about
140 mg of protein) in PBS, it was dissolved in 27 ml of the cell
dissolution buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA,
1 mM Pefabloc, 10 .mu.g/ml), disrupted by sonication at 60 W for 30
minutes, followed by the addition of 3 ml of the cell dissolution
buffer containing 10% SB-12 and further sonication twice at 60 W
for 30 minutes, and the final concentration was made 150 mM in 5 M
NaCl. It was centrifuged to separate the supernatant, filtered with
a 0.45 .mu.m filter to prepare a sample for FPLC (Amersham
Pharmacia Biotech).
[0199] Chromatography
[0200] An anion exchange chromatography was performed using the
HiLoad 16/10 Q Sepharose High Performance (Amersham Pharmacia
Biotech) and eluted at a flow rate of 2 ml/min with a salt
concentration gradient of 0 M to 0.5 M NaCl. The starting buffer
was 20 mM Tris-HCl, pH 7.5, 0 M NaCl, 1 mM EDTA, 0.1% CHAPS, and
the elution buffer was the starting buffer+0.5 M NaCl. From each of
the eluted fractions 20 .mu.l was aliquoted and was subjected to a
west western blot using GST-WT3, and the fraction (FIG. 3) for
which the WT1 interacting protein was detected was used for the
subsequent purification.
[0201] Using the same buffer, an anion exchange chromatography was
performed using MONO Q HR 5/5 (Amersham Pharmacia Biotech). The
flow rate was 1 ml/min. A similar assay was performed for each
fraction (FIG. 4). A hydrophobic interaction chromatography was
performed using Phenyl Superose HR 5/5 (Amersham Pharmacia
Biotech), and then was eluted at a flow rate of 0.5 ml/min and a
salt concentration gradient of 1.0 M to 0 M. The starting buffer
was 20 mM Tris-HCl, pH 7.5, 1.0 M (NH4)2SO4, 1 mM EDTA, and the
elution buffer was 20 mM Tris-HCl, pH 7.5, 0 M (NH4).sub.2SO4, 1 mM
EDTA. Assay of each fraction was performed by west western blot as
in the above, and from the fraction in which the WT1 interacting
protein was detected (FIG. 5), protein was recovered with
trichloroacetic acid, separated on SDS-PAGE (5.0% gel), transferred
to a PVDF membrane (proBlott, Applied Biosystem), and was used for
peptide mapping.
[0202] Thus, as after hydrophobic interaction chromatography, a
specific band was separated at around 115 kDa on SDS-PAGE, this was
transferred to a PVDF membrane, subjected to CBB staining and west
western blot to identify a CBB band (FIG. 6). The bands was excised
from the PVDF membrane, and was directly used for peptide
mapping.
[0203] Peptide Mapping
[0204] The protein transferred to the PVDF membrane was decomposed
with lysil endopeptidase on the membrane, and after the formed
peptide was separated on HPLC, the molecular weight of each peptide
was determined by a mass spectrometer (MALDI-TOF MS). The molecular
weight pattern of peptides was compared to the amino acid sequence
database to predict the original protein.
[0205] Identification of WTIP
[0206] As a result of peptide mapping, the presence of three
different proteins was predicted in the band: HYPA/FGP11 (accession
No. AF049528), RNA helicase associated protein (AF083255), and siah
binding protein 1 (U51586). The base sequence at the 3'-end has not
been determined for HYPA/FBP11, the base sequence at the 5'-end has
not been determined for sia binding protein, and the entire
sequence of the RNA helicase associated protein has been
determined, but the molecular weight was 77.9 kDa which was
significantly different from that predicted based on the position
of the band. In order to identify the WT1 interacting protein from
among them, a his-tagged protein of the known region of each gene
was constructed and its binding to GST-WT3 was investigated. As a
result, since HYPA/FBP11 bound to GST-WT3, the cloning of the
full-length and the identification of the binding site were
attempted.
[0207] The construction of the above his-tagged protein was
performed as follows:
[0208] Using cDNA of human leukemia cell line K562 or KG-1 as the
template, and the initiation codon of a primer (HYPA/FBP11
(AF049523)) to which a XhoI recognition sequence was added at the
3'-end as 1, PCR was performed for 1-1218 (amino acids 1-406),
1-534 (amino acids 1-178), 1-294 (amino acids 1-98) bases to
construct insertion fragments (B in FIG. 7), which were inserted
into pET-21b(+) (Novagen) that adds a his tag to the C-terminal. In
almost the same manner as for the GST fusion protein, E. coli was
cultured, and the cells were recovered. However, pT-Trx was not
used. The cells derived from 1 ml of the culture were dissolved in
100 .mu.l of the sample buffer for SDS-PAGE, and were directly used
for west western blot and Western blot. The antibody used in
Western blot was anti-His tag antibody (c-term, Invitrogen).
Example 2
Cloning of cDNA Encoding WTIP
[0209] WTIP has a full length of about 3 kbp, and about 1.3 kbp at
the 5'-end is HYPA/FBP11. This contains the WW domain that is known
to bind to a proline-rich domain. Since the full-length of a mouse
homolog of FBP11 was reported, cDNA derived from K562 and from KG-1
was amplified using the sequence at the 3'-end, and an about 3 kbp
fragment was obtained.
[0210] Using the cDNA of K562, PCR and RACE (RApid Amplification of
cDNA ends) were performed from the known sequences. First, for the
5'-end, 5' RACE was performed using a base sequence
5'-CTTTGCTGGTTGGCTCTCCTCCTCTTCT-3' (SEQ ID NO: 9) in a known region
as the antisense primer to confirm the 5'-end of the known
sequence.
[0211] For the 3'-end, using a base sequence
5'-TTGATCATCATCCAGTTGCTCCAAAAGGG-3' (SEQ ID NO: 10) corresponding
to the C-terminal of the mouse FBP11 translation region as the
antisense primer and a base sequence
5'-TGGGACAAATGCCTGGAATGATGTCGTC-3' (SEQ ID NO: 11) in HYPA as the
sense primer, PCR was performed to determine the base sequence, and
it was found that this region contained NY-REN-6.
[0212] Furthermore, using a base sequence
5'-CAGCGATCAGAGTCTCGTTCTGCTTCAG-3' (SEQ ID NO: 12) in the NY-REN-6
region as the sense primer, 3' RACE was performed to determine the
base sequence. By combining these fragments, the full-length of
WTIP was cloned.
[0213] An about 3 kbp DNA fragment obtained as above encodes the
full-length of human WTIP, and the base sequence is shown in SEQ ID
NO: 1 and the deduced amino acid sequence in SEQ ID NO: 2. The
determination of the base sequence was performed by the TA cloning
(Invitrogen) of cDNA. For DNA database search, Basic BLAST (BLAST
2.0) on the NCBI server was used. For the analysis of the base
sequence, GENETYX-Mac 10.1 (SDC Software Development Institute) was
used.
[0214] The structure of full-length WITP is shown in FIG. 7.
Example 3
WT1 Interaction Site for WTIP
[0215] The WW domain is known to bind to a prolin-rich domain
having the Pro-Pro-Leu-Pro (PPLP) (SEQ ID NO: 15) motif, but the
same motif cannot be found in Huntingtin. Though WT1 also has the
proline-rich domain, it is not a PPLP (SEQ ID NO: 15) motif. In
order to investigate whether WT1 is bound to WTIP via the WW
domain, west western blot was performed using a his-tagged protein
of a WTIP mutant. As shown in FIG. 8, a mutant containing no WW
domain had lost the ability of binding with GST-WT3, indicating
that the WW domain is required for the binding of WTIP and WT1.
Example 4
Reactivity of WT1 and WTIP in the cell
[0216] Using a cell line U937 in which WT1 was forcefully
expressed, a co-precipitation experiment by, immunoprecipitation
was performed to confirm the binding of WT1 and WTIP in the
cell.
[0217] 1.times.107 cells were dissolved in 500 .mu.l of the ELB
buffer (50 mM HEPES, pH 7.5, 250 mM NaCl, 0.5 mM EDTA, 0.1% NP-40,
1 mM Pefabloc (TM), Complete (TM) EDTA free, 1 mM DTT), disrupted
by sonication, allowed to stand on ice for 1 hour, and centrifuged
at 15000 rpm, 4.degree. C. to recover the supernatant. To the
supernatant was added 4 .mu.g of anti-WT1 antibody (C-19,
Santa-Cruz), gently stirred at 4.degree. C. for 2 hours, and then
20 .mu.l (bed volume) of Protein A Sepharose (Amersham Pharmacia
Biotech) was added, and was further stirred for 2 hours. After
washing the Protein A Sepharose 3 times with 1 mL of the ELB
buffer, 40 O. of the sample buffer for SDS-PAGE was added. The
reaction supernatant was recovered as the flow through, to which
the sample buffer was similarly added. The two were heated to
90.degree. C. for 5 minutes, and was subjected to Western blot.
After electrophoresis on a 7.5% polyacrylamide gel, it was
transferred to Immobilon P (Millipore), and was subjected to color
development with NBT/BCIP using anti-WTIP antibody as the primary
antibody and anti-rabbit IgG antibody (alkaline
phosphatase-conjugated, Promega) as the second antibody.
[0218] In FIG. 9, FT represents flow through and IP represents the
binding fraction of Protein A Sepharose. At the position of the
arrowhead in FIG. 9, a band of WTIP was observed. Thus, it was
shown that WT1 and WTIP bind in the cells in which WT1 was
forcefully expressed.
Sequence CWU 1
1
15 1 2979 DNA Homo sapiens Nucleotide sequence encoding human WTIP
CDS (14)..(2884) 1 tctgagcccg acg atg agg ccg ggg acg gga gct gag
cgt gga ggc ctc 49 Met Arg Pro Gly Thr Gly Ala Glu Arg Gly Gly Leu
1 5 10 atg gtg agt gaa atg gag agc cat cct ccc tcg cag ggt cct ggg
gac 97 Met Val Ser Glu Met Glu Ser His Pro Pro Ser Gln Gly Pro Gly
Asp 15 20 25 ggg gag cgg aga ttg tcc ggc tca agc ctc tgc tcc ggc
tct tgg gtc 145 Gly Glu Arg Arg Leu Ser Gly Ser Ser Leu Cys Ser Gly
Ser Trp Val 30 35 40 tct gct gac ggc ttc ctg agg aga cgg ccc tcg
atg ggg cac cct ggc 193 Ser Ala Asp Gly Phe Leu Arg Arg Arg Pro Ser
Met Gly His Pro Gly 45 50 55 60 atg cat tat gcc cca atg gga atg cac
cct atg ggt cag aga gcg aat 241 Met His Tyr Ala Pro Met Gly Met His
Pro Met Gly Gln Arg Ala Asn 65 70 75 atg cct cct gta cct cat gga
atg atg ccg cag atg atg ccc cct atg 289 Met Pro Pro Val Pro His Gly
Met Met Pro Gln Met Met Pro Pro Met 80 85 90 gga ggg cca cca atg
gga caa atg cct gga atg atg tcg tca gta atg 337 Gly Gly Pro Pro Met
Gly Gln Met Pro Gly Met Met Ser Ser Val Met 95 100 105 cct gga atg
atg atg tct cat atg tct cag gct tcc atg cag cct gcc 385 Pro Gly Met
Met Met Ser His Met Ser Gln Ala Ser Met Gln Pro Ala 110 115 120 tta
ccg cca gga gta aat agt atg gat gta gca gca ggt aca gca tct 433 Leu
Pro Pro Gly Val Asn Ser Met Asp Val Ala Ala Gly Thr Ala Ser 125 130
135 140 ggt gca aaa tca atg tgg act gaa cat aaa tca cct gat gga agg
act 481 Gly Ala Lys Ser Met Trp Thr Glu His Lys Ser Pro Asp Gly Arg
Thr 145 150 155 tac tac tac aac act gaa acc aaa cag tct acc tgg gag
aaa cca gat 529 Tyr Tyr Tyr Asn Thr Glu Thr Lys Gln Ser Thr Trp Glu
Lys Pro Asp 160 165 170 gat ctt aaa aca cct gct gag caa ctc tta tct
aaa tgc ccc tgg aag 577 Asp Leu Lys Thr Pro Ala Glu Gln Leu Leu Ser
Lys Cys Pro Trp Lys 175 180 185 gaa tac aaa tca gat tct gga aag cct
tac tat tat aat tct caa aca 625 Glu Tyr Lys Ser Asp Ser Gly Lys Pro
Tyr Tyr Tyr Asn Ser Gln Thr 190 195 200 aaa gaa tct cgc tgg gcc aaa
cct aaa gaa ctt gag gat ctt gaa gga 673 Lys Glu Ser Arg Trp Ala Lys
Pro Lys Glu Leu Glu Asp Leu Glu Gly 205 210 215 220 tac cag aat acc
att gtt gct gga agt ctt att aca aaa tca aac ctg 721 Tyr Gln Asn Thr
Ile Val Ala Gly Ser Leu Ile Thr Lys Ser Asn Leu 225 230 235 cat gca
atg atc aaa gct gaa gaa agc agt aag caa gaa gag tgc acc 769 His Ala
Met Ile Lys Ala Glu Glu Ser Ser Lys Gln Glu Glu Cys Thr 240 245 250
aca aca tca aca gcc cca gtc cct aca aca gaa att ccg acc aca atg 817
Thr Thr Ser Thr Ala Pro Val Pro Thr Thr Glu Ile Pro Thr Thr Met 255
260 265 agc acc atg gct gct gcc gaa gca gca gct gct gtt gtt gca gca
gca 865 Ser Thr Met Ala Ala Ala Glu Ala Ala Ala Ala Val Val Ala Ala
Ala 270 275 280 gca gcg gca gca gca gca gca gct gca gcc aat gct aat
gct tcc act 913 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Asn Ala Asn
Ala Ser Thr 285 290 295 300 tct gct tct aat act gtc agt gga act gtt
cca gtt gtt cct gag cct 961 Ser Ala Ser Asn Thr Val Ser Gly Thr Val
Pro Val Val Pro Glu Pro 305 310 315 gaa gtt act tcc att gtt gct act
gtt gta gat aat gag aat aca gta 1009 Glu Val Thr Ser Ile Val Ala
Thr Val Val Asp Asn Glu Asn Thr Val 320 325 330 act att tca act gag
gaa caa gca caa ctt act agt acc cct gct att 1057 Thr Ile Ser Thr
Glu Glu Gln Ala Gln Leu Thr Ser Thr Pro Ala Ile 335 340 345 cag gat
caa agt gtg gaa gta tcc agt aat act gga gaa gaa aca tct 1105 Gln
Asp Gln Ser Val Glu Val Ser Ser Asn Thr Gly Glu Glu Thr Ser 350 355
360 aag caa gaa act gta gct gat ttt act ccc aaa aaa gaa gag gag gag
1153 Lys Gln Glu Thr Val Ala Asp Phe Thr Pro Lys Lys Glu Glu Glu
Glu 365 370 375 380 agc caa cca gca aag aaa aca tac act tgg aat aca
aag gaa gag gca 1201 Ser Gln Pro Ala Lys Lys Thr Tyr Thr Trp Asn
Thr Lys Glu Glu Ala 385 390 395 aag caa gct ttt aaa gaa tta ttg aaa
gaa aag cgg gta cca tcg aat 1249 Lys Gln Ala Phe Lys Glu Leu Leu
Lys Glu Lys Arg Val Pro Ser Asn 400 405 410 gct tca tgg gag cag gct
atg aaa atg att att aat gat cca cga tac 1297 Ala Ser Trp Glu Gln
Ala Met Lys Met Ile Ile Asn Asp Pro Arg Tyr 415 420 425 agt gct ttg
gca aag tta agt gaa aaa aag caa gcc ttt aat gcc tat 1345 Ser Ala
Leu Ala Lys Leu Ser Glu Lys Lys Gln Ala Phe Asn Ala Tyr 430 435 440
aaa gtc cag aca gaa aaa gaa gaa aaa gaa gaa gca aga tca aag tac
1393 Lys Val Gln Thr Glu Lys Glu Glu Lys Glu Glu Ala Arg Ser Lys
Tyr 445 450 455 460 aaa gag gct aag gaa tcc ttt cag cgt ttt ctt gaa
aat cat gag aaa 1441 Lys Glu Ala Lys Glu Ser Phe Gln Arg Phe Leu
Glu Asn His Glu Lys 465 470 475 atg act tct aca acc aga tac aaa aaa
gca gag caa atg ttt gga gag 1489 Met Thr Ser Thr Thr Arg Tyr Lys
Lys Ala Glu Gln Met Phe Gly Glu 480 485 490 atg gaa gtt tgg aat gca
ata tca gaa cgt gat cgt ctt gaa atc tat 1537 Met Glu Val Trp Asn
Ala Ile Ser Glu Arg Asp Arg Leu Glu Ile Tyr 495 500 505 gaa gat gtt
ttg ttc ttt ctt tca aaa aaa gaa aag gaa caa gca aag 1585 Glu Asp
Val Leu Phe Phe Leu Ser Lys Lys Glu Lys Glu Gln Ala Lys 510 515 520
cag ttg cga aag aga aat tgg gaa gcc tta aaa aac ata ctt gac aac
1633 Gln Leu Arg Lys Arg Asn Trp Glu Ala Leu Lys Asn Ile Leu Asp
Asn 525 530 535 540 atg gct aat gta aca tac tct acc act tgg tct gaa
gcc cag cag tat 1681 Met Ala Asn Val Thr Tyr Ser Thr Thr Trp Ser
Glu Ala Gln Gln Tyr 545 550 555 ctg atg gat aat cca act ttt gca gaa
gat gag gag tta caa aat atg 1729 Leu Met Asp Asn Pro Thr Phe Ala
Glu Asp Glu Glu Leu Gln Asn Met 560 565 570 gac aaa gaa gat gca tta
att tgc ttt gaa gaa cac att cgg gct tta 1777 Asp Lys Glu Asp Ala
Leu Ile Cys Phe Glu Glu His Ile Arg Ala Leu 575 580 585 gaa aag gag
gaa gaa gaa gaa aaa cag aag agt ttg ctg aga gaa agg 1825 Glu Lys
Glu Glu Glu Glu Glu Lys Gln Lys Ser Leu Leu Arg Glu Arg 590 595 600
aga cga cag cga aaa aat agg gaa tct ttc cag ata ttt tta gat gaa
1873 Arg Arg Gln Arg Lys Asn Arg Glu Ser Phe Gln Ile Phe Leu Asp
Glu 605 610 615 620 tta cat gaa cat gga caa ctg cat tct atg tca tct
tgg atg gaa ttg 1921 Leu His Glu His Gly Gln Leu His Ser Met Ser
Ser Trp Met Glu Leu 625 630 635 tat cca act att agt tct gat att aga
ttc act aat atg ctt ggt cag 1969 Tyr Pro Thr Ile Ser Ser Asp Ile
Arg Phe Thr Asn Met Leu Gly Gln 640 645 650 cct gga tca act gca ctt
gat ctt ttc aag ttt tat gtt gag gat ctt 2017 Pro Gly Ser Thr Ala
Leu Asp Leu Phe Lys Phe Tyr Val Glu Asp Leu 655 660 665 aaa gca cgt
tat cat gac gag aag aag ata ata aaa gac att cta aag 2065 Lys Ala
Arg Tyr His Asp Glu Lys Lys Ile Ile Lys Asp Ile Leu Lys 670 675 680
gat aaa gga ttt gta gtt gaa gta aac act act ttt gaa gat ttt gtg
2113 Asp Lys Gly Phe Val Val Glu Val Asn Thr Thr Phe Glu Asp Phe
Val 685 690 695 700 gcg ata atc agt tca act aaa aga tca act aca tta
gat gct gga aat 2161 Ala Ile Ile Ser Ser Thr Lys Arg Ser Thr Thr
Leu Asp Ala Gly Asn 705 710 715 atc aaa ttg gct ttc aat agt tta cta
gaa aag gca gaa gcc cgt gaa 2209 Ile Lys Leu Ala Phe Asn Ser Leu
Leu Glu Lys Ala Glu Ala Arg Glu 720 725 730 cgt gaa aga gaa aaa gaa
gag gct cgg aag atg aaa cga aaa gaa tct 2257 Arg Glu Arg Glu Lys
Glu Glu Ala Arg Lys Met Lys Arg Lys Glu Ser 735 740 745 gca ttt aag
agt atg tta aaa caa gct gct cct ccg ata gaa ttg gat 2305 Ala Phe
Lys Ser Met Leu Lys Gln Ala Ala Pro Pro Ile Glu Leu Asp 750 755 760
gct gtc tgg gaa gat atc cgt gag aga ttt gta aaa gag cca gca ttt
2353 Ala Val Trp Glu Asp Ile Arg Glu Arg Phe Val Lys Glu Pro Ala
Phe 765 770 775 780 gag gac ata act cta gaa tct gaa aga aaa cga ata
ttt aaa gat ttt 2401 Glu Asp Ile Thr Leu Glu Ser Glu Arg Lys Arg
Ile Phe Lys Asp Phe 785 790 795 atg cat gtg ctt gag cat gaa tgt cag
cat cat cat tca aag aac aag 2449 Met His Val Leu Glu His Glu Cys
Gln His His His Ser Lys Asn Lys 800 805 810 aaa cat tct aag aaa tct
aaa aaa cat cat agg aaa cgt tcc cgc tct 2497 Lys His Ser Lys Lys
Ser Lys Lys His His Arg Lys Arg Ser Arg Ser 815 820 825 cga tcg ggg
tca gat tca gat gat gat gat agc cat tca aag aaa aaa 2545 Arg Ser
Gly Ser Asp Ser Asp Asp Asp Asp Ser His Ser Lys Lys Lys 830 835 840
aga cag cga tca gag tct cgt tct gct tca gaa cat tct tct agt gca
2593 Arg Gln Arg Ser Glu Ser Arg Ser Ala Ser Glu His Ser Ser Ser
Ala 845 850 855 860 gag tct gag aga agt tat aaa aag tca aaa aag cat
aag aag aaa agt 2641 Glu Ser Glu Arg Ser Tyr Lys Lys Ser Lys Lys
His Lys Lys Lys Ser 865 870 875 aag aag agg aga cat aaa tct gac tct
cca gaa tcc gat gct gag cga 2689 Lys Lys Arg Arg His Lys Ser Asp
Ser Pro Glu Ser Asp Ala Glu Arg 880 885 890 gag aag gat aaa aaa gaa
aaa gat cgg gaa agt gaa aaa gac aga act 2737 Glu Lys Asp Lys Lys
Glu Lys Asp Arg Glu Ser Glu Lys Asp Arg Thr 895 900 905 aga caa aga
tca gaa tca aaa cac aaa tcg cct aag aaa aag act gga 2785 Arg Gln
Arg Ser Glu Ser Lys His Lys Ser Pro Lys Lys Lys Thr Gly 910 915 920
aag gat tct ggt aat tgg gat act tct ggc agc gaa ctg agt gaa ggg
2833 Lys Asp Ser Gly Asn Trp Asp Thr Ser Gly Ser Glu Leu Ser Glu
Gly 925 930 935 940 gaa ttg gaa aag cgc aga aga acc ctt ttg gag caa
ctg gat gat gat 2881 Glu Leu Glu Lys Arg Arg Arg Thr Leu Leu Glu
Gln Leu Asp Asp Asp 945 950 955 caa taaattatac caaatatatg
tttacagtat gatttaaagt ctgattcaga 2934 Gln ccagggactc tattttaaag
ttcaactgaa ataacactgg gaaaa 2979 2 957 PRT Homo sapiens Amino aicd
sequence human WTIP 2 Met Arg Pro Gly Thr Gly Ala Glu Arg Gly Gly
Leu Met Val Ser Glu 1 5 10 15 Met Glu Ser His Pro Pro Ser Gln Gly
Pro Gly Asp Gly Glu Arg Arg 20 25 30 Leu Ser Gly Ser Ser Leu Cys
Ser Gly Ser Trp Val Ser Ala Asp Gly 35 40 45 Phe Leu Arg Arg Arg
Pro Ser Met Gly His Pro Gly Met His Tyr Ala 50 55 60 Pro Met Gly
Met His Pro Met Gly Gln Arg Ala Asn Met Pro Pro Val 65 70 75 80 Pro
His Gly Met Met Pro Gln Met Met Pro Pro Met Gly Gly Pro Pro 85 90
95 Met Gly Gln Met Pro Gly Met Met Ser Ser Val Met Pro Gly Met Met
100 105 110 Met Ser His Met Ser Gln Ala Ser Met Gln Pro Ala Leu Pro
Pro Gly 115 120 125 Val Asn Ser Met Asp Val Ala Ala Gly Thr Ala Ser
Gly Ala Lys Ser 130 135 140 Met Trp Thr Glu His Lys Ser Pro Asp Gly
Arg Thr Tyr Tyr Tyr Asn 145 150 155 160 Thr Glu Thr Lys Gln Ser Thr
Trp Glu Lys Pro Asp Asp Leu Lys Thr 165 170 175 Pro Ala Glu Gln Leu
Leu Ser Lys Cys Pro Trp Lys Glu Tyr Lys Ser 180 185 190 Asp Ser Gly
Lys Pro Tyr Tyr Tyr Asn Ser Gln Thr Lys Glu Ser Arg 195 200 205 Trp
Ala Lys Pro Lys Glu Leu Glu Asp Leu Glu Gly Tyr Gln Asn Thr 210 215
220 Ile Val Ala Gly Ser Leu Ile Thr Lys Ser Asn Leu His Ala Met Ile
225 230 235 240 Lys Ala Glu Glu Ser Ser Lys Gln Glu Glu Cys Thr Thr
Thr Ser Thr 245 250 255 Ala Pro Val Pro Thr Thr Glu Ile Pro Thr Thr
Met Ser Thr Met Ala 260 265 270 Ala Ala Glu Ala Ala Ala Ala Val Val
Ala Ala Ala Ala Ala Ala Ala 275 280 285 Ala Ala Ala Ala Ala Ala Asn
Ala Asn Ala Ser Thr Ser Ala Ser Asn 290 295 300 Thr Val Ser Gly Thr
Val Pro Val Val Pro Glu Pro Glu Val Thr Ser 305 310 315 320 Ile Val
Ala Thr Val Val Asp Asn Glu Asn Thr Val Thr Ile Ser Thr 325 330 335
Glu Glu Gln Ala Gln Leu Thr Ser Thr Pro Ala Ile Gln Asp Gln Ser 340
345 350 Val Glu Val Ser Ser Asn Thr Gly Glu Glu Thr Ser Lys Gln Glu
Thr 355 360 365 Val Ala Asp Phe Thr Pro Lys Lys Glu Glu Glu Glu Ser
Gln Pro Ala 370 375 380 Lys Lys Thr Tyr Thr Trp Asn Thr Lys Glu Glu
Ala Lys Gln Ala Phe 385 390 395 400 Lys Glu Leu Leu Lys Glu Lys Arg
Val Pro Ser Asn Ala Ser Trp Glu 405 410 415 Gln Ala Met Lys Met Ile
Ile Asn Asp Pro Arg Tyr Ser Ala Leu Ala 420 425 430 Lys Leu Ser Glu
Lys Lys Gln Ala Phe Asn Ala Tyr Lys Val Gln Thr 435 440 445 Glu Lys
Glu Glu Lys Glu Glu Ala Arg Ser Lys Tyr Lys Glu Ala Lys 450 455 460
Glu Ser Phe Gln Arg Phe Leu Glu Asn His Glu Lys Met Thr Ser Thr 465
470 475 480 Thr Arg Tyr Lys Lys Ala Glu Gln Met Phe Gly Glu Met Glu
Val Trp 485 490 495 Asn Ala Ile Ser Glu Arg Asp Arg Leu Glu Ile Tyr
Glu Asp Val Leu 500 505 510 Phe Phe Leu Ser Lys Lys Glu Lys Glu Gln
Ala Lys Gln Leu Arg Lys 515 520 525 Arg Asn Trp Glu Ala Leu Lys Asn
Ile Leu Asp Asn Met Ala Asn Val 530 535 540 Thr Tyr Ser Thr Thr Trp
Ser Glu Ala Gln Gln Tyr Leu Met Asp Asn 545 550 555 560 Pro Thr Phe
Ala Glu Asp Glu Glu Leu Gln Asn Met Asp Lys Glu Asp 565 570 575 Ala
Leu Ile Cys Phe Glu Glu His Ile Arg Ala Leu Glu Lys Glu Glu 580 585
590 Glu Glu Glu Lys Gln Lys Ser Leu Leu Arg Glu Arg Arg Arg Gln Arg
595 600 605 Lys Asn Arg Glu Ser Phe Gln Ile Phe Leu Asp Glu Leu His
Glu His 610 615 620 Gly Gln Leu His Ser Met Ser Ser Trp Met Glu Leu
Tyr Pro Thr Ile 625 630 635 640 Ser Ser Asp Ile Arg Phe Thr Asn Met
Leu Gly Gln Pro Gly Ser Thr 645 650 655 Ala Leu Asp Leu Phe Lys Phe
Tyr Val Glu Asp Leu Lys Ala Arg Tyr 660 665 670 His Asp Glu Lys Lys
Ile Ile Lys Asp Ile Leu Lys Asp Lys Gly Phe 675 680 685 Val Val Glu
Val Asn Thr Thr Phe Glu Asp Phe Val Ala Ile Ile Ser 690 695 700 Ser
Thr Lys Arg Ser Thr Thr Leu Asp Ala Gly Asn Ile Lys Leu Ala 705 710
715 720 Phe Asn Ser Leu Leu Glu Lys Ala Glu Ala Arg Glu Arg Glu Arg
Glu 725 730 735 Lys Glu Glu Ala Arg Lys Met Lys Arg Lys Glu Ser Ala
Phe Lys Ser 740 745 750 Met Leu Lys Gln Ala Ala Pro Pro Ile Glu Leu
Asp Ala Val Trp Glu 755 760 765 Asp Ile Arg Glu Arg Phe Val Lys Glu
Pro Ala Phe Glu Asp Ile Thr 770 775 780 Leu Glu Ser Glu Arg Lys Arg
Ile Phe Lys Asp Phe Met His Val Leu 785 790 795 800 Glu His Glu Cys
Gln His His His Ser Lys Asn Lys Lys His Ser Lys 805 810 815 Lys Ser
Lys Lys His His Arg Lys Arg Ser Arg Ser Arg Ser Gly Ser 820 825 830
Asp Ser Asp Asp Asp Asp Ser His Ser Lys Lys Lys Arg Gln Arg Ser 835
840 845 Glu Ser Arg Ser Ala Ser Glu His Ser Ser Ser Ala Glu Ser Glu
Arg 850 855 860 Ser Tyr
Lys Lys Ser Lys Lys His Lys Lys Lys Ser Lys Lys Arg Arg 865 870 875
880 His Lys Ser Asp Ser Pro Glu Ser Asp Ala Glu Arg Glu Lys Asp Lys
885 890 895 Lys Glu Lys Asp Arg Glu Ser Glu Lys Asp Arg Thr Arg Gln
Arg Ser 900 905 910 Glu Ser Lys His Lys Ser Pro Lys Lys Lys Thr Gly
Lys Asp Ser Gly 915 920 925 Asn Trp Asp Thr Ser Gly Ser Glu Leu Ser
Glu Gly Glu Leu Glu Lys 930 935 940 Arg Arg Arg Thr Leu Leu Glu Gln
Leu Asp Asp Asp Gln 945 950 955 3 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 3 ttgaattcaa tgggctccga
cgtgcgg 27 4 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 4 ttgtcgacca tgggatcctc atgctt 26 5 27 DNA
Artificial Sequence Description of Artificial Sequence Primer 5
ttgaattcaa tgggctccga cgtgcgg 27 6 26 DNA Artificial Sequence
Description of Artificial Sequence Primer 6 ttgtcgacga agacaccgtg
cgtgtg 26 7 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 7 ttgaattcag atccaatggg ccagcag 27 8 26 DNA
Artificial Sequence Description of Artificial Sequence Primer 8
ttgtcgacga agacaccgtg cgtgtg 26 9 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 9 ctttgctggt tggctctcct
cctcttct 28 10 29 DNA Artificial Sequence Description of Artificial
Sequence Primer 10 ttgatcatca tccagttgct ccaaaaggg 29 11 28 DNA
Artificial Sequence Description of Artificial Sequence Primer 11
tgggacaaat gcctggaatg atgtcgtc 28 12 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 12 cagcgatcag agtctcgttc
tgcttcag 28 13 6 PRT Artificial Sequence Description of Artificial
Sequence 6X His tag 13 His His His His His His 1 5 14 10 PRT
Artificial Sequence Description of Artificial Sequence 10X His tag
14 His His His His His His His His His His 1 5 10 15 4 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
motif 15 Pro Pro Leu Pro 1
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