U.S. patent application number 12/325631 was filed with the patent office on 2009-04-23 for novel hemopoietin receptor protein, nr12.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha, a corporation of Japan. Invention is credited to Masatsugu Maeda, Noriko Yaguchi.
Application Number | 20090105459 12/325631 |
Document ID | / |
Family ID | 26550628 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090105459 |
Kind Code |
A1 |
Maeda; Masatsugu ; et
al. |
April 23, 2009 |
NOVEL HEMOPOIETIN RECEPTOR PROTEIN, NR12
Abstract
A novel hemopoietin receptor gene (NR12) was successfully
isolated by extracting motifs conserved among the amino acid
sequences of known hemopoietin receptors and by using the predicted
sequence. The NR12 gene encodes two forms of proteins, a
transmembrane type and a soluble type. The expression of the NR12
gene was detected in tissues containing hematopoietic cells. NR12
is a novel hemopoietin receptor molecule involved in the regulation
of immune system and hematopoiesis in vivo. Thus, NR12 is useful in
the search for novel hematopoietic factors that functionally bind
to the NR12 receptor, and in the development of therapeutic drugs
for diseases associated with immunity or hematopoiesis.
Inventors: |
Maeda; Masatsugu; (Ibaraki,
JP) ; Yaguchi; Noriko; (Ibaraki, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Chugai Seiyaku Kabushiki Kaisha, a
corporation of Japan
|
Family ID: |
26550628 |
Appl. No.: |
12/325631 |
Filed: |
December 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12205799 |
Sep 5, 2008 |
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12325631 |
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12205753 |
Sep 5, 2008 |
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12205799 |
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11595320 |
Nov 9, 2006 |
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12205753 |
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11274375 |
Nov 14, 2005 |
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11595320 |
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10105930 |
Mar 25, 2002 |
7045595 |
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11274375 |
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PCT/JP00/06654 |
Sep 27, 2000 |
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10105930 |
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11274375 |
Nov 14, 2005 |
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11595320 |
Nov 9, 2006 |
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10105930 |
Mar 25, 2002 |
7045595 |
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11274375 |
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PCT/JP00/06654 |
Sep 27, 2000 |
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10105930 |
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Current U.S.
Class: |
530/387.3 ;
530/387.9 |
Current CPC
Class: |
C07K 14/715 20130101;
A61P 37/02 20180101; C07K 16/2866 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.9 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 1999 |
JP |
11/273358 |
Aug 3, 2000 |
JP |
2000-240397 |
Claims
1. A purified antibody or antigen-binding fragment thereof that
binds specifically to a polypeptide consisting of the amino acid
sequence of SEQ ID NO:8.
2. A purified antibody or fragment thereof that binds specifically
to a polypeptide consisting of the amino acid sequence from Gly at
position 24 to Ser at position 428 of SEQ ID NO:8.
3. The antibody or fragment thereof of claim 1, wherein the
antibody is a monoclonal antibody.
4. The antibody or fragment thereof of claim 2, wherein the
antibody is a monoclonal antibody.
5. The antibody or fragment thereof of claim 1, wherein the
antibody is a chimeric antibody.
6. The antibody or fragment thereof of claim 2, wherein the
antibody is a chimeric antibody.
7. The antibody or fragment thereof of claim 1, wherein the
antibody is a humanized antibody.
8. The antibody or fragment thereof of claim 2, wherein the
antibody is a humanized antibody.
9. The antibody or fragment thereof of claim 1, wherein the
antibody is a human antibody.
10. The antibody or fragment thereof of claim 2, wherein the
antibody is a human antibody.
11. A modified antibody or antigen-binding fragment thereof that
binds specifically to a polypeptide consisting of the amino acid
sequence of SEQ ID NO:8, wherein the antibody or fragment thereof
is bound to a molecule using chemical modification.
12. A modified antibody or antigen-binding fragment thereof that
binds specifically to a polypeptide consisting of the amino acid
sequence from Gly at position 24 to Ser at position 428 of SEQ ID
NO:8, wherein the antibody or fragment thereof is bound to a
molecule using chemical modification.
13. The antibody fragment of claim 1, wherein the antibody fragment
is a single chain Fv.
14. The antibody fragment of claim 2, wherein the antibody fragment
is a single chain Fv.
15. A method of forming an immune complex, the method comprising
the steps of: (a) providing the antibody or fragment thereof of
claim 1, and (b) contacting the antibody or fragment thereof with a
polypeptide that binds to the antibody.
16. A method of forming an immune complex, the method comprising
the steps of: (a) providing the antibody or fragment thereof of
claim 2, and (b) contacting the antibody or fragment thereof with a
polypeptide that binds to the antibody.
17. A method of forming an immune complex, the method comprising
the steps of: (a) providing the antibody or fragment thereof of
claim 5, and (b) contacting the antibody or fragment thereof with a
polypeptide that binds to the antibody.
18. A method of forming an immune complex, the method comprising
the steps of: (a) providing the antibody or fragment thereof of
claim 6, and (b) contacting the antibody or fragment thereof with a
polypeptide that binds to the antibody.
19. The method of claim 15, further comprising the step of: (c)
detecting the immune complex.
20. The method of claim 16, further comprising the step of: (c)
detecting the immune complex.
21. The method of claim 17, further comprising the step of: (c)
detecting the immune complex.
22. The method of claim 18, further comprising the step of: (c)
detecting the immune complex.
Description
[0001] This application is a divisional of U.S. application Ser.
Nos. 12/205,799 and 12/205,753, both filed Sep. 5, 2008, each being
a divisional of U.S. application Ser. No. 11/595,320, filed Nov. 9,
2006, which is a continuation of U.S. application Ser. No.
11/274,375, filed Nov. 14, 2005, now abandoned, which is a
divisional of U.S. application Ser. No. 10/105,930, filed Mar. 25,
2002, now U.S. Pat. No. 7,045,595, which is a continuation-in-part
of International Patent Application No. PCT/JP00/06654, filed Sep.
27, 2000, which claims the benefit of Japanese Patent Application
Nos. 11-273358, filed Sep. 27, 1999, and 2000-240397, filed Aug. 3,
2000. Each of the prior U.S. applications is herein incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates to novel hemopoietin receptor
proteins, and genes encoding them, as well as methods for producing
and using the same.
BACKGROUND
[0003] A large number of cytokines are known as humoral factors
that regulate proliferation/differentiation of various cells, or
that regulate the maintenance, activation, and death of
differentiated mature cells. There are specific receptors for these
cytokines, which are categorized into several families based on
their structural similarities (Hilton D. J., in "Guidebook to
Cytokines and Their Receptors" edited by Nicola N. A. (A Sambrook
& Tooze Publication at Oxford University Press), 1994, p
8-16).
[0004] On the other hand, as compared to the similarities of their
receptors, the homology of the primary-structure among cytokines is
quite low. No significant amino acid homology has be observed, even
among cytokine members that belong to the same receptor family.
This explains the functional specificity of respective cytokines,
as well as similarities among cellular reactions induced by each
cytokine.
[0005] Representative examples of the above-mentioned receptor
families are the tyrosine kinase receptor family, hemopoietin
receptor family, tumor necrosis factor (TNF) receptor family, and
transforming growth factor (TGF) receptor family. Different signal
transduction pathways have been reported to be involved with each
of these families. Among these receptor families, many receptors of
the hemopoietin receptor family in particular are expressed in
blood cells and immunocytes, and their ligands, cytokines, are
often termed as hemopoietic factors or interleukins. Some of these
hemopoietic factors or interleukins exist within blood and are
thought to be involved in systemic humoral regulation of
hemopoietic or immune functions.
[0006] This contrasts with the belief that cytokines belonging to
other families are often involved in only topical regulation. Some
of these hemopoietins can be taken as hormone-like factors, and
representative peptide hormones, such as the growth hormone,
prolactin, or leptin receptors, also belong to the hemopoietin
receptor family. Because of these hormone-like systemic regulatory
features, it is anticipated that administration of these
hemopoietins can be applied to the treatment of various diseases.
Among the large number of cytokines known, those that are presently
being clinically applied include erythropoietin, G-CSF, GM-CSF, and
IL-2. Combined with IL-11, LIF, and IL-12 that are currently under
consideration for clinical trials, and the above-mentioned peptide
hormones, such as the growth hormone and prolactin, it can be
envisaged that by searching novel cytokines that bind to
hemopoietin receptors among the above-mentioned various receptor
superfamilies, it is possible to find a cytokine that can be
clinically applied with a higher efficiency.
[0007] As mentioned above, cytokine receptors have structural
similarities among the family members. Using these similarities,
many investigations are aimed at finding novel receptors. In
particular, many receptors of the tyrosine kinase receptor family
have already been cloned, using its highly conserved sequence at
the catalytic site (Matthews et al., Cell 65(7):143-52, 1991). In
comparison, hemopoietin receptors do not have a tyrosine
kinase-like enzyme activity domain in their cytoplasmic regions,
and their signal transductions are known to be mediated through
associations with other tyrosine kinase proteins existing freely in
the cytoplasm. Though the sites on receptors binding with these
cytoplasmic tyrosine kinases, called JAK kinases group, are
conserved among family members, the homology is not very high
(Murakami et al., Proc. Natl. Acad. Sci. USA 88:11349-11353, 1991).
Actually, the sequence that best characterizes these hemopoietin
receptors exists in the extracellular region. In particular, a five
amino acid motif, Trp-Ser-Xaa-Trp-Ser (wherein "Xaa" is an
arbitrary amino acid; SEQ ID NO:21), is conserved in almost all of
the hemopoietin receptors. Therefore, novel receptors may be
obtained by searching for novel family members using this motif
sequence. In fact, these approaches have already led to the
identification of the IL-11 receptor (Robb et al., J. Biol. Chem.
271(23):13754-13761, 1996), the leptin receptor (Gainsford et al.,
Proc. Natl. Acad. Sci. USA 93(25):14564-8, 1996), and the IL-13
receptor (Hilton et al., Proc. Natl. Acad. Sci. USA 93(1):497-501,
1996).
SUMMARY
[0008] The present invention provides novel hemopoietin receptor
proteins, and DNA encoding these proteins. The present invention
also provides a vector into which the DNA has been inserted, a
transformant harboring the DNA, and a method for producing
recombinant proteins using the transformant. The present invention
also provides methods of screening for compounds that bind to the
protein.
[0009] Initially, the inventors attempted to find a novel receptor
using oligonucleotides encoding the Trp-Ser-Xaa-Trp-Ser motif (WS
motif; SEQ ID NO:21) as the probe by the plaque hybridization
method, RT-PCR method, and so on. However, it was extremely
difficult to strictly select only those to which all 15 nucleotides
that encode the motif would completely hybridize under the usual
hybridization conditions, because the oligonucleotide
"tggag(t/c)nnntggag(t/c)" (wherein "n" is an arbitrary nucleotide;
SEQ ID NO:22) encoding the motif was short, having just 15 base
pairs. Further, because the g/c content of the oligonucleotide was
high, higher than usual annealing temperature conditions were
required to strictly select those sequences in which all the 15
nucleotides hybridized completely to the oligonucleotide.
Therefore, performing screening under normal hybridization
experiment conditions was extremely difficult.
[0010] To solve these problems, the inventors searched for
additional motifs, other than the site of the above-mentioned WS
motif that is conserved in the hemopoietin receptor family. The
inventors found that a residue, either tyrosine or histidine,
located 13 to 27 amino acids upstream of the WS motif in the
extracellular region was highly conserved in the receptor family.
Furthermore, additional search for consensus sequences that are
frequently found in the 6 amino acids from the above Tyr/H is
residue toward the C-terminus led to the identification of the
following consensus sequence:
(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg
(hereinafter, abbreviated as the YR motif). However, this YR motif
is not exactly a perfect consensus sequence, and the combination of
the nucleotide sequences that encode the motif is very complicated.
Therefore, it is practically impossible to synthesize and provide
oligonucleotides that encode all of the amino acid sequences as
probes for hybridization, which is a practical method for
screening, or as primers aimed for RT-PCR.
[0011] Accordingly, the inventors looked for other approaches to
practically search for novel members of the hemopoietin receptor
family using the above two motifs as probes, and determined that it
would be appropriate to perform a database search on the computer
using partial amino acid sequences of known hemopoietin receptors,
including both motifs as the query. The inventors repeated TblastN
searches on the gss and htgs database in GenBank, using partial
amino acid sequences from multiple known hemopoietin receptors as
the query. As a result, many positive clones, including known
hemopoietin receptors, were obtained in all cases. Next, the
nucleotide sequence around those sequences which seemed to be
positive at a high rate was converted to the amino acid sequence.
Genes considered to encode members of the receptor family were
selected by BlastX search, in which the amino acid sequences
(converted from the nucleotide sequences of the clones) were
compared to those of known hemopoietin receptors. According to the
two-step Blast search above, human genome sequences encoding two
clones of known hemopoietin receptor genes and one clone of novel
hemopoietin receptor gene were identified. Subsequently, specific
oligonucleotide primers were designed based on the exon sequences
predicted from the obtained nucleotide sequence. Clones
corresponding to the N-terminal region and C-terminal region of
NR12 were obtained by conducting 5'-RACE and 3'-RACE methods using
the primers, and cDNA libraries of human fetal liver, adult thymus,
and adult testis as the templates. The complete nucleotide sequence
of the full-length cDNA was revealed by determining the nucleotide
sequences of both clones, and connecting the sequence at the
duplicated center region.
[0012] From structural analyses, at least three kinds of
transcription products derived from splice variants were
recognized. A cDNA clone of these splice variants comprising 337
amino acids and potentially encoding a secretory form soluble
receptor protein was named NR12.1; the other two clones, comprising
428 amino acids and 629 amino acids respectively and each encoding
transmembrane form receptor proteins, were named NR12.2 and NR12.3.
Because repeated structure of cysteine residues, YR motif, WS
motif, and so on, that are conserved in the extracellular region of
other family members were well conserved in the primary structure
of all the isolated cDNA clones of NR12, it was considered that
these clones encode typical hemopoietin receptors.
[0013] Subsequently, RT-PCR was performed using primer sets
specific to NR12.1, NR12.2, and NR12.3, respectively, against mRNA
derived from various human tissue. Then, tissues expressing these
genes were searched, and the distribution and the expression
pattern of the genes in each human tissue were analyzed. Finally,
in order to discard the possibility of non-specific amplification
and to quantify the amount of the RT-PCR products, the products of
RT-PCR were subjected to Southern blotting using cDNA fragments
specific to the respective clones. The result indicated that these
clones are mainly expressed in hematopoietic cell line tissue and
immune cell line tissue.
[0014] Furthermore, the present inventors succeeded in obtaining
two clones (NR12.4 and NR12.5) encoding complete proteins that were
3 amino acids different from NR12.2 and NR12.3, respectively, by
conducting PCR cloning against the cDNA library of human thymus
(wherein five clones generically named "NR12" were isolated).
[0015] Based on the above features of NR12, NR12 is presumed to be
a novel hemopoietin receptor molecule related to the regulation of
the immune system or hematopoiesis. The gene encoding NR12 will be
extremely useful in the screening for novel hematopoietic factors
that can functionally bind to the receptor.
[0016] Moreover, the present inventors succeeded in isolating
genomic fragments of mouse receptor homologues by conducting
xenogenic cross hybridization cloning using cDNA of human NR12 as
the probe. It is expected that further elucidation of the in vivo
function of the receptor protein is possible by constructing mutant
mouse lacking NR12 gene using the mouse gene fragments.
[0017] Consequently, the present invention relates to novel
hemopoietin receptors and genes encoding the receptors, as well as
use of the same. More specifically, the present invention provides
the following:
[0018] (1) a DNA selected from the group consisting of:
[0019] (a) a DNA encoding a protein comprising the amino acid
sequence of any one of SEQ ID NOs:2, 4, 6, 8, and 10;
[0020] (b) a DNA comprising the coding region of the nucleotide
sequence of any one of SEQ ID NOs:1, 3, 5, 7, and 9;
[0021] (c) a DNA encoding a protein comprising the amino acid
sequence of any one of SEQ ID NOs:2, 4, 6, 8, and 10, in which one
or more amino acids are modified by substitution, deletion,
insertion, and/or addition, wherein said protein is functionally
equivalent to the protein consisting of the amino acid sequence of
any of SEQ ID NOs:2, 4, 6, 8, and 10; and,
[0022] (d) a DNA hybridizing under stringent conditions with a DNA
consisting of the nucleotide sequence of any one of SEQ ID NOs:1,
3, 5, 7, and 9, and encoding a protein that is functionally
equivalent to the protein consisting of the amino acid sequence of
any one of SEQ ID NOs:2, 4, 6, 8, and 10;
[0023] (2) a DNA encoding a partial peptide of a protein consisting
of the amino acid sequence of any one of SEQ ID NOs:2, 4, 6, 8, and
10;
[0024] (3) a protein or peptide that is encoded by the DNA
described in (1) or (2);
[0025] (4) a vector into which the DNA described in (1) or (2) is
inserted;
[0026] (5) a transformant harboring the DNA described in (1) or
(2), or the vector described in (4);
[0027] (6) a method for producing the protein or peptide of (3),
comprising the steps of: culturing said transformant of (5), and
recovering the expressed protein from said transformant or the
culture supernatant;
[0028] (7) an antibody binding to the protein of (3);
[0029] (8) a polynucleotide complementary to either a DNA that
comprises the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5,
7, and 9 or its complementary strand, wherein the polynucleotide
comprises at least 15 nucleotides; and,
[0030] (9) a method of screening for a compound that binds to the
protein of (3), comprising the steps of:
[0031] (a) contacting a test sample with said protein or partial
peptide thereof;
[0032] (b) detecting the binding activity of the test sample with
the protein or partial peptide thereof; and,
[0033] (c) selecting the compound that binds to the protein or
partial peptide thereof.
[0034] The present invention provides a novel hemopoietin receptor
"NR12". According to the results of the database searches on
GenBank as well as 5'-RACE and 3'-RACE analysis, the present
inventors finally succeeded in identifying and isolating a novel
hemopoietin receptor gene NR12. It was found that at least three
splice variants are transcribed from NR12. One of these variants,
the cDNA clone NR12.1, encodes a soluble receptor-like protein. The
other two predicted to encode transmembrane receptor proteins, cDNA
clone NR12.2 and NR12.3, encode a protein presumed to have an
intracellular region as short as 51 amino acids and as long as 252
amino acids, respectively.
[0035] Furthermore, the present inventors conducted PCR cloning
against a cDNA library of human thymus to isolate the continuous
full-length coding sequences (CDS). A clone having almost the same
full-length ORF as NR12.2 was named NR12.4, and that having almost
the same full-length ORF as NR12.3 was named NR12.5.
[0036] The nucleotide sequence of NR12.1 cDNA is shown in SEQ ID
NO:1, and the corresponding amino acid sequence of the protein
encoded by the cDNA is shown in SEQ ID NO:2. The nucleotide
sequence of NR12.2 cDNA is shown in SEQ ID NO:3, and the
corresponding amino acid sequence of the protein encoded by the
cDNA is shown in SEQ ID NO:4. The nucleotide sequence of NR12.3
cDNA is shown in SEQ ID NO:5, and the corresponding amino acid
sequence of the protein encoded by the cDNA is shown in SEQ ID
NO:6. The nucleotide sequence of NR12.4 cDNA is shown in SEQ ID
NO:7, and the corresponding amino acid sequence of the protein
encoded by the cDNA is shown in SEQ ID NO:8. The nucleotide
sequence of NR12.5 cDNA is shown in SEQ ID NO:9, and the
corresponding amino acid sequence of the protein encoded by the
cDNA is shown in SEQ ID NO:10.
[0037] Because the extracellular regions of NR12.1, NR12.2, NR12.3,
NR12.4, and NR12.5 are almost identical, these regions are thought
to have the same tertiary structure and thereby recognize the same
specific ligand.
[0038] Analyses of the gene expression in various human organs
using RT-PCR revealed: strong expression of NR12 in hematopoietic
cell line tissues and immune cell line tissues such as adult
spleen, thymus, lymph node, bone marrow, and peripheral leukocyte;
and expression in testis, liver, lung, kidney, pancreas, and
gastrointestinal tract, such as small intestine and colon.
Additionally, expression of NR12 was also observed in all the
analyzed mRNA derived from human fetal organs. From the revealed
distribution pattern of NR12 gene expression, it was presumed that
NR12 encodes a novel hematopoietic factor receptor, primarily
because localization of strong expression in tissues thought to
include immune cell lines and hematopoietic cells was detected.
Furthermore, the fact that NR12 expression was observed in tissues
other than those described above suggests that NR12 can regulate
not only physiological functions of the immune system and
hematopoietic system in vivo but also various other physiological
functions in vivo.
[0039] The above NR12 proteins are potentially useful for medical
application. Since NR12.1 is expressed in thymus, peripheral
leukocytes, and spleen, it is predicted to be a receptor for an
unknown hemopoietic factor. Therefore, NR12 proteins are useful
tools in the identification of the unknown hemopoietic factor. They
may also be used to screen a peptide library or synthetic chemical
compounds to isolate or identify agonists and antagonists that can
functionally bind to the NR12 molecule. Moreover, clinical
application is expected of novel molecules binding to the NR12
molecule and specific antibodies that can limit the function of the
NR12 molecule to regulate the immune response or hematopoiesis in
vivo, by searching such molecules and antibodies.
[0040] NR12 is expected to be expressed in a restricted population
of cells in the hemopoietic tissues, and thus, anti-NR12 antibodies
are useful for the isolation of such cell populations. The isolated
cell populations may be used in cell transplantation. Furthermore,
it is expected that the anti-NR12 antibody may be used for the
diagnosis or treatment of diseases, such as leukemia.
[0041] On the other hand, the soluble proteins comprising the
extracellular domain of NR12 protein and the splice variant of
NR12, NR12.1, may be used as a decoy-type receptor to inhibit the
NR12 ligand. They may be useful for the treatment of diseases in
which NR12 is implicated, such as leukemia.
[0042] The present invention includes proteins that are
functionally equivalent to the NR12 protein. For example,
homologues of human NR12 protein are included. Herein, the term
"functionally equivalent" refers to proteins having an equivalent
biological activity as compared to that of an NR12 protein. Such
biological activity may include the protein activity as a membrane
bound or soluble form hematopoietic factor receptor.
[0043] Methods of introducing mutations for preparing proteins that
are functionally equivalent to another protein are well known to a
person skilled in the art. For example, one skilled in the art may
use site-directed mutagenesis (Hashimoto-Gotoh et al., Gene
152:271-275, 1995; Zoller et al., Methods Enzymol. 100:468-500,
1983; Kramer et al., Nucleic Acids Res. 12:9441-9456, 1984; Kramer
et al., Methods. Enzymol. 154:350-367, 1987; Kunkel, Proc. Natl.
Acad. Sci. USA 82:488-492, 1985; Kunkel, Methods Enzymol.
85:2763-2766, 1988) and such in order to introduce an appropriate
mutation into the amino acid sequence of the human NR12 protein and
prepare a protein that is functionally equivalent to the protein.
Mutation of amino acids may occur in nature as well. The proteins
of the present invention includes proteins having the amino acid
sequence of human NR12 protein in which one or more amino acids are
mutated, so long as the resulting proteins are functionally
equivalent to human NR12 protein.
[0044] As a protein functionally equivalent to the NR12 protein of
the invention, the following can be specifically mentioned: one in
which one or two, preferably, two to 30, more preferably, two to 10
amino acids are deleted in any one of the amino acid sequences of
SEQ ID NOs:2, 4, 6, 8, or 10; one in which one or two, preferably,
two to 30, more preferably, two to 10 amino acids have been added
into any one of the amino acid sequences of SEQ ID NOs:2, 4, 6, 8,
or 10; one in which one or two, preferably, two to 30, more
preferably, two to 10 amino acids have been substituted with other
amino acids in any one of the amino acid sequences of SEQ ID NOs:2,
4, 6, 8, or 10.
[0045] As for the amino acid residue to be mutated, it is
preferable that it be mutated into a different amino acid that
allows the properties of the amino acid side-chain to be conserved.
Examples of properties of amino acid side chains are the following:
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), and amino acids
comprising the following side chains: an aliphatic side-chain (G,
A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a
sulfur atom containing side-chain (C, M); a carboxylic acid and
amide containing side-chain (D, N, E, Q); a base containing
side-chain (R, K, H); and an aromatic containing side-chain (H, F,
Y, W) (The parenthetic letters indicate the one-letter codes of
amino acids).
[0046] It is known that a protein may have an amino acid sequence
modified by deletion, addition, and/or substitution of other amino
acids for one or more amino acid residues, yet still retain its
biological activity (Mark et al., Proc. Natl. Acad. Sci. USA
81:5662-5666, 1984; Zoller et al., Nucleic Acids Res. 10:6487-6500,
1982; Wang et al., Science 224:1431-1433; Dalbadie-McFarland et
al., Proc. Natl. Acad. Sci. USA 79:6409-6413, 1982).
[0047] A fusion protein comprising human NR12 protein is an example
of a protein in which one or more amino acids residues have been
added to the amino acid sequence of a human NR12 protein (e.g., SEQ
ID NO:2, 4 6, 8 or 10). A fusion protein is made by fusing the
human NR12 protein with another peptide(s) or protein(s) and is
included in the present invention. A fusion protein can be prepared
by ligating a DNA encoding the human NR12 protein of the present
invention with a DNA encoding another peptide(s) or protein(s) in
frame, introducing the ligated DNA into an expression vector, and
expressing the fusion gene in a host. Methods known by one skilled
in the art can be used for preparing such a fusion gene. There is
no restriction as to the other peptide(s) or protein(s) that is
(are) fused to the protein of the present invention.
[0048] Other peptide(s) to be fused with a protein of the present
invention include known peptides, for example, FLAG (Hopp et al.,
Biotechnology 6:1204-1210, 1988), 6.times.His consisting of six His
(histidine) residues, 10.times.His, Influenza agglutinin (HA),
human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag,
HSV-tag, E-tag, SV40T antigen fragment, lck tag, .alpha.-tubulin
fragment, B-tag, Protein C fragment, and so on. Other examples of
proteins to be fused with the protein of the present invention are
the GST (glutathione-S-transferase), Influenza agglutinin (HA),
immunoglobulin constant region, .beta.-galactosidase, MBP
(maltose-binding protein), and such.
[0049] Fusion proteins can be prepared by fusing commercially
available DNA encoding these peptides or proteins with DNA encoding
a protein of the present invention and expressing the fused DNA
prepared.
[0050] The hybridization technique (Sambrook et al., Molecular
Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) is
well known to those skilled in the art as an alternative method for
preparing a protein functionally equivalent to a certain protein.
More specifically, one skilled in the art can utilize the general
procedure to obtain a protein functionally equivalent to a human
NR12 protein by isolating DNA having a high homology with the whole
or part of a DNA sequence encoding the human NR12 protein (e.g.,
SEQ ID NO:1, 3, 5, 7 or 9). Thus, the proteins of the present
invention include such proteins, that are encoded by DNAs that
hybridizes with a DNA encoding a human NR12 protein or part thereof
and that are functionally equivalent to a human NR12 protein.
Examples include homologues of human NR12 in other mammals (for
example, those of monkey, rat, mouse, rabbit, and bovine gene). In
order to isolate a cDNA with high homology to a DNA encoding a
human NR12 protein from animals, it is preferable to use a
hematopoietic cell line tissue such as spleen, thymus, lymph node,
bone marrow, and peripheral leukocyte; however, the invention is
not limited thereto.
[0051] Stringent hybridization conditions for isolating DNA
encoding proteins functionally equivalent to a human NR12 protein
can be suitably selected by one skilled in the art, and for
example, low-stringent conditions can be given. Low-stringent
conditions are, for example, 42.degree. C., 2.times.SSC, and 0.1%
SDS, and preferably, 50.degree. C., 2.times.SSC, and 0.1% SDS. High
stringent conditions are more preferable and include, for example,
65.degree. C., 2.times.SSC, and 0.1% SDS. Under these conditions,
at lower temperatures, the DNA obtained will have a lower homology.
Conversely, it is expected that the homology of the obtained DNA
will be higher at higher temperatures. However, several factors
other than temperature, such as salt concentration, can also
influence the stringency of hybridization and one skilled in the
art can routinely select the factors to accomplish a similar
stringency.
[0052] In place of hybridization, the gene amplification method,
for example, the polymerase chain reaction (PCR) method can be
utilized to isolate the object DNA, using primers synthesized based
on the sequence information of a DNA (e.g., SEQ ID NO:1, 3, 5, 7 or
9) encoding human NR12 protein.
[0053] Proteins that are functionally equivalent to human NR12
protein, encoded by DNA isolated through the above hybridization
technique or by the gene amplification techniques, usually have a
high homology to the amino acid sequence of the human NR12 protein.
The proteins of the present invention also include proteins that
are functionally equivalent to the human NR12 protein, which also
have a high homology with the protein comprising any one of the
amino acid sequences of SEQ ID NO:2, 4, 6, 8, and 10. High homology
is normally defined as a homology of 70% or higher, preferably 80%
or higher, more preferably 90% or higher, and most preferably 95%
or higher. The homology of a protein can be determined by the
algorithm in "Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad.
Sci. USA (1983) 80, 726-730".
[0054] The amino acid sequence, molecular weight, isoelectric
point, the presence or absence of sugar chains, and the form of a
protein of the present invention may differ according to the
producing cells, host, or purification method described below.
However, so long as the obtained protein has an equivalent function
to human NR12 protein (SEQ ID NO:2, 4, 6, 8 or 10), it is included
in the present invention. For example, if a protein of the present
invention is expressed in prokaryotic cells, such as E. coli, a
methionine residue is added at the N-terminus of the amino acid
sequence of the expressed protein. If a protein of the present
invention is expressed in eukaryotic cells, such as mammalian
cells, the N-terminal signal sequence is removed. Such proteins are
also included as proteins of the present invention.
[0055] For example, as a result of analysis of the protein of the
invention based on the method in "Von Heijne, G., Nucleic Acids
Research, (1986), 14, 4683-4690", it was presumed that the signal
sequence extends from the 1.sup.st Met to the 23.sup.rd Gly in the
amino acid sequences of SEQ ID NO:2, 4, 6, 8 and 10. Therefore, the
present invention encompasses a protein comprising the sequence
from the 24.sup.th Gly to 337.sup.th Cys in the amino acid sequence
of SEQ ID NO:2. Similarly, the present invention encompasses a
protein comprising the sequence from the 24.sup.th Gly to
428.sup.th Ser in the amino acid sequence of SEQ ID NO:4.
Similarly, the present invention encompasses a protein comprising
the sequence from the 24.sup.th Gly to 629.sup.th Lys in the amino
acid sequence of SEQ ID NO:6. Similarly, the present invention
encompasses a protein comprising the sequence from the 24.sup.th
Gly to 428.sup.th Ser in the amino acid sequence of SEQ ID NO:8.
Similarly, the present invention encompasses a protein comprising
the sequence from the 24.sup.th Gly to 629.sup.th Lys in the amino
acid sequence of SEQ ID NO:10.
[0056] The term "substantially pure" as used herein in reference to
a given polypeptide means that the polypeptide is substantially
free from other biological macromolecules. For example, the
substantially pure polypeptide is at least 75%, 80, 85, 95, or 99%
pure by dry weight. Purity can be measured by any appropriate
standard method known in the art, for example, by column
chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0057] Accordingly, the invention includes a polypeptide having a
sequence shown as SEQ ID NO:2, 4, 6, 8 or 10. The invention also
includes a polypeptide, or fragment thereof, that differs from the
corresponding sequence shown as SEQ ID NO:2, 4, 6, 8 or 10. The
differences are, preferably, differences or changes at a
non-essential residue or a conservative substitution. In one
embodiment, the polypeptide includes an amino acid sequence at
least about 60% identical to a sequence shown as SEQ ID NO:2, 4, 6,
8 or 10, or a fragment thereof. Preferably, the polypeptide is at
least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical
to SEQ ID NO:2, 4, 6, 8 or 10 and has at least one receptor
activity described herein, e.g., a hemopoietin receptor activity.
Preferred polypeptide fragments of the invention are at least 10%,
preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the
length of the sequence shown as SEQ ID NO:2, 4, 6, 8 or 10 and have
at least one receptor activity described herein, e.g., a
hemopoietin receptor activity. Or alternatively, the fragment can
be merely an immunogenic fragment.
[0058] A protein of the present invention can be prepared by
methods known to one skilled in the art, as a recombinant protein,
and also as a natural protein. A recombinant DNA can be prepared by
inserting a DNA encoding the protein of the present invention (for
example, the DNA comprising the nucleotide sequence of SEQ ID NO:1,
3, 5, 7 or 9) into a suitable expression vector, introducing the
vector into a suitable host cell, and collecting the extract from
the resulting transformant. After obtaining the extract,
recombinant protein can be purified and prepared by subjecting to
chromatography, such as ion exchange chromatography, reverse phase
chromatography, gel filtration, and such, or affinity
chromatography, wherein antibodies against the protein of the
present invention are immobilized, or using one or more of these
columns in combination.
[0059] Further, when a protein of the present invention is
expressed within host cells (for example, animal cells and E.
coli), as a fusion protein with glutathione-S-transferase protein
or as a recombinant protein supplemented with multiple histidines,
the expressed recombinant protein can be purified using a
glutathione column or nickel column.
[0060] After purifying the fusion protein, it is also possible to
exclude regions other than the objective protein by cutting with
thrombin, factor-Xa, and such, as required.
[0061] A natural protein may be isolated by methods known to one
skilled in the art. For example, extracts of tissue or cells
expressing a protein of the invention may be reacted with an
affinity column described below, to which antibodies binding to the
human NR12 protein are attached, to isolate the natural protein.
Polyclonal or monoclonal antibodies may be used.
[0062] The present invention also includes partial peptides of the
proteins of the present invention. A partial peptide consists of an
amino acid sequence specific to a protein of the present invention
and is composed of at least 7 amino acids, preferably more than 8
amino acids, and more preferably more than 9 amino acids. The
partial peptides may be useful, for example, for preparing
antibodies against a protein of the present invention; for
screening compounds binding to a protein of the present invention,
or for screening accelerators or inhibitors of a protein of the
present invention. Alternatively, they may be used as antagonists
for the ligand of a protein of the present invention. A partial
peptide of a protein of the present invention is, for example, a
partial peptide having the active center of the protein consisting
of the amino acid sequences of SEQ ID NO:2, 4, 6, 8, or 10.
Additionally, the partial peptides may comprise one or more regions
of the hydrophilic region and hydrophobic region determined by
hydrophobicity plot analysis. These partial peptides may contain
the whole hydrophilic region or a part of a hydrophilic region, or
may contain the whole or a part of the hydrophobic region.
Moreover, for example, soluble proteins and proteins comprising
extracellular regions of a protein of the invention are also
encompassed in the invention.
[0063] The partial peptides of the invention may be produced by
genetic engineering techniques, well-known peptide synthesizing
methods, or by excising a protein of the invention with a suitable
peptidase. For example, the solid phase synthesizing method or
liquid phase synthesizing method may be used as peptide
synthesizing method.
[0064] Another object of the present invention is to provide a DNA
encoding a protein of the present invention. The DNA may be useful
for producing the above proteins of the present invention in vivo
or in vitro. Furthermore, for example, it is also possible to use
the DNA for application to gene therapy and such of diseases
arising from abnormalities of the gene encoding the protein of the
present invention. The DNA may be provided in any form, so long as
it encodes a protein of the present invention. Thus, the DNA may be
a cDNA synthesized from mRNA, genomic DNA, or chemically
synthesized DNA. Furthermore, a DNA comprising any nucleotide
sequence based on the degeneracy of genetic code may be included so
long as it encodes a protein of the present invention.
[0065] As used herein, an "isolated nucleic acid" is a nucleic
acid, the structure of which is not identical to that of any
naturally occurring nucleic acid or to that of any fragment of a
naturally occurring genomic nucleic acid spanning more than three
genes. The term therefore covers, for example, (a) a DNA which has
the sequence of part of a naturally occurring genomic DNA molecule
but is not flanked by both of the coding sequences that flank that
part of the molecule in the genome of the organism in which it
naturally occurs; (b) a nucleic acid incorporated into a vector or
into the genomic DNA of a prokaryote or eukaryote in a manner such
that the resulting molecule is not identical to any naturally
occurring vector or genomic DNA; (c) a separate molecule such as a
cDNA, a genomic fragment, a fragment produced by polymerase chain
reaction (PCR), or a restriction fragment; and (d) a recombinant
nucleotide sequence that is part of a hybrid gene, i.e., a gene
encoding a fusion protein. Specifically excluded from this
definition are nucleic acids present in random, uncharacterized
mixtures of different DNA molecules, transfected cells, or cell
clones, e.g., as these occur in a DNA library such as a cDNA or
genomic DNA library.
[0066] Accordingly, in one aspect, the invention provides an
isolated or purified nucleic acid molecule that encodes a
polypeptide described herein or a fragment thereof. Preferably, the
isolated nucleic acid molecule includes a nucleotide sequence that
is at least 60% identical to the nucleotide sequence shown in SEQ
ID NO:1, 3, 5, 7 or 9. More preferably, the isolated nucleic acid
molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide
sequence shown in SEQ ID NO:1, 3, 5, 7 or 9. In the case of an
isolated nucleic acid molecule which is longer than or equivalent
in length to the reference sequence, e.g., SEQ ID NO:1, 3, 5, 7 or
9, the comparison is made with the full length of the reference
sequence. Where the isolated nucleic acid molecule is shorter that
the reference sequence, e.g., shorter than SEQ ID NO:1, 3, 5, 7 or
9, the comparison is made to a segment of the reference sequence of
the same length (excluding any loop required by the homology
calculation).
[0067] As used herein, "% identity" of two amino acid sequences, or
of two nucleic acid sequences, is determined using the algorithm of
Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268,
1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci.
USA 90:5873-5877, 1993). Such an algorithm is incorporated into the
NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol.
215:403-410, 1990). BLAST nucleotide searches are performed with
the NBLAST program, score=100, wordlength=12. BLAST protein
searches are performed with the XBLAST program, score=50,
wordlength=3. To obtain gapped alignment for comparison purposes
GappedBLAST is utilized as described in Altschul et al. (Nucleic
Acids Res. 25:3389-3402, 1997). When utilizing BLAST and
GappedBLAST programs the default parameters of the respective
programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide
sequences homologous to a nucleic acid molecule of the
invention.
[0068] The DNA of the present invention can be prepared by any
method known to a person skilled in the art. For example, the DNA
of the present invention may be prepared by constructing a cDNA
library from cells expressing the protein of the present invention,
and conducting hybridization using as a probe a partial sequence of
a DNA of the present invention (for example, SEQ ID NO:1, 3, 5, 7
or 9). A cDNA library may be constructed, for example, according to
the method described in the literature (Sambrook et al., Molecular
Cloning, Cold Spring Harbor Laboratory Press, 1989), or a
commercial cDNA library may be used. Alternatively, the DNA may be
prepared by obtaining RNA from a cell expressing a protein of the
present invention, synthesizing oligo DNA based on the sequence of
a DNA of the present invention (for example, SEQ ID NO:1, 3, 5, 7
or 9), conducting PCR using the synthesized DNA as primers, and
amplifying the cDNA encoding a protein of the present
invention.
[0069] By determining the nucleotide sequence of the obtained cDNA,
the translation region encoded by the cDNA can be determined, and
the amino acid sequence of the protein of the present invention can
be obtained. Furthermore, genomic DNA can be isolated by screening
genomic DNA libraries using the obtained cDNA as a probe.
[0070] Specifically, this can be done as follows: first, mRNA is
isolated from cells, tissues, or organs expressing a protein of the
invention (for example, hematopoietic-competent cell line tissue
such as spleen, thymus, lymph node, bone marrow, peripheral
leukocyte and immunocompetent cell line tissue, and such). To
isolate the mRNA, at first, whole RNA is prepared using well-known
methods, for example, the guanidine ultracentrifugation method
(Chirgwin et al., Biochemistry 18:5294-5299, 1979), the AGPC method
(Chomczynski et al., Anal. Biochem. 162:156-159, 1987), and such.
Next, mRNA from whole mRNA can be purified using the mRNA
Purification Kit (Pharmacia), and such. Alternatively, mRNA may be
directly prepared by QuickPrep mRNA Purification Kit
(Pharmacia).
[0071] cDNA can then be synthesized using reverse transcriptase
from the obtained mRNA. cDNA can be synthesized by using the AMV
Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku
Kogyo), etc. Additionally, cDNA synthesis and amplification may be
also performed using the primer and such described herein,
following the 5'-RACE method (Frohman et al. Proc. Natl. Acad. Sci.
USA 85:8998-9002, 1988; Belyavsky et al., Nucleic Acids Res.
17:2919-2932, 1989) utilizing the polymerase chain reaction (PCR)
and the 5'-Ampli FINDER RACE Kit (Clontech).
[0072] The objective DNA fragment is prepared from the obtained PCR
product and ligated with a vector DNA. Thus, a recombinant vector
is created and introduced into E. coli, and such, and colonies are
selected to prepare the desired recombinant vector. The nucleotide
sequence of the objective DNA can be verified by conventional
methods, for example, dideoxynucleotide chain termination.
[0073] With regards to the DNA of the invention, a sequence with
higher expression efficiency can be designed by considering the
codon usage frequency in the host used for the expression (Grantham
et al., Nucleic Acids Res. 9:43-74, 1981). The DNA of the present
invention may also be modified using commercially available kits
and conventional methods. Illustrative modifications include, for
instance, digestion by restriction enzymes, insertion of synthetic
oligonucleotides and suitable DNA fragments, addition of linkers,
insertion of a initiation codon (ATG) and/or stop codon (TAA, TGA,
or TAG), and such.
[0074] Specifically, the DNA of the present invention includes DNA
comprising the nucleotide sequence from the 98.sup.th "A" to the
1108.sup.th "C" of SEQ ID NO:1; the 98.sup.th "A" to 1381.sup.st
"C" of SEQ ID NO:3; the 98.sup.th "A" to 1984.sup.th "G" of SEQ ID
NO:5; the 1.sup.st "A" to 1284.sup.th "C" of SEQ ID NO:7; and the
1.sup.st "A" to 1887.sup.th "G" of SEQ ID NO:9.
[0075] Furthermore, the present invention includes DNA that
hybridize under stringent conditions to the DNA consisting of any
one of the nucleotide sequence of SEQ ID NO:1, 3, 5, 7 or 9, so
long as the resulting DNA encodes a protein functionally equivalent
to the above-mentioned protein of the invention.
[0076] One skilled in the art can suitably select stringent
conditions, and for example, low-stringent conditions can be given.
Low-stringent conditions are, 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 preferable are highly stringent
conditions which are, for example, 65.degree. C., 2.times.SSC, and
0.1% SDS. Under these conditions, the higher the temperature, the
higher the homology of the obtained DNA will be. The above
hybridizing DNA is preferably a natural DNA, such as cDNA and
chromosomal DNA.
[0077] Moreover, the present invention provides a vector containing
a DNA of the present invention as an insert. The vector of the
present invention may be useful for maintaining the DNA of the
present invention in host cells or producing the protein of the
present invention.
[0078] If the host cell is E. coli (such as JM109, DH5.alpha.,
HB101, and XL1Blue), any vector may be used as long as it contains
the "ori" for amplification in E. coli that enables large-scale
preparation, and a selection marker for transformants (for example,
a drug-resistance gene that enables selection by a drug such as
ampicillin, tetracycline, kanamycin, and chloramphenicol). For
example, M13-series vectors, pUC-series vectors, pBR322,
pBluescript, pCR-Script, and so on can be used. For the purpose of
subcloning or excision of a cDNA, pGEM-T, pDIRECT, pT7, and such
may be used as well. For producing the protein of the present
invention, an expression vector is especially useful. For example,
if the protein is to be expressed in E. coli, the expression vector
must have characteristics such as those mentioned above to be
amplified in E. coli. Additionally, when E. coli, such as JM109,
DH5.alpha., HB101, or XL1 Blue, is used as the host cell, the
vector must have a promoter, for example, the lacZ promoter (Ward
et al., Nature 341:544-546, 1989; FASEB J. 6:2422-2427, 1992), the
araB promoter (Better et al., Science 240:1041-1043, 1988), the T7
promoter, and such, that can efficiently express the desired gene
in E. coli. Such vectors include pGFX-5X-1 (Pharmacia), "QIAexpress
system" (Qiagen), pEGFP, pET (in this case, a host is preferably
BL21 which expresses T7 RNA polymerase), and so on, except those
mentioned above.
[0079] Vectors may be introduced into host cells, for example, by
the calcium chloride method or electroporation. The vector may also
contain a signal sequence for polypeptide secretion. The pe1B
signal sequence (Lei et al., J. Bacteriol. 169:4379, 1987) may be
used to produce the proteins in the periplasm in E. coli.
[0080] For example, an expression vector for the preparation of a
protein of the present invention may be a mammal-derived expression
vector (for example, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids.
Res. 18(17):5322, 1990), pEF, and pCDM8); an insect cell-derived
expression vector (for example, "Bac-to-BAC baculovirus expression
system" (GIBCO BRL), pBacPAK8); a plant-derived expression vector
(for example, pMH1 and pMH2); an animal virus-derived expression
vector (for example, pHSV, pMV, and pAdexLcw); a retrovirus-derived
expression vector (for example, pZIpneo); an yeast-derived
expression vector (for example, "Pichia Expression Kit"
(Invitrogen), pNV11, and SP-Q01); or a Bacillus subtilis-derived
expression vectors (for example, pPL608 and pKTH50), other than E.
coli.
[0081] For the expression in animal cells, such as CHO, COS, and
NIH3T3 cells, the expression vector must have a promoter such as
the SV40 promoter (Mulligan et al., Nature 277:108, 1979), MMLV-LTR
promoter, the EF1.alpha. promoter (Mizushima et al., Nucleic Acids
Res. 18:5322, 1990), and the CMV promoter. More preferably, the
vector may contain a marker gene for the selection of transformants
(for example, a drug resistance gene for selection by a drug such
as neomycin and G418). Such vectors include pMAM, pDR2, pBK-RSV,
pBK-CMV, pOPRSV, pOp13, and so on.
[0082] Furthermore, in order to achieve stable gene expression and
amplification of the copy number of genes in cell, CHO cells
deficient in the metabolic pathway for nucleotide synthesis may be
used. The CHO cell is transfected with an expression vector
comprising the DHFR gene that complements the deficiency (for
example, pCHO I), then the vector may be amplified by methotrexate
(MTX) treatment. For transient gene expression, COS cells
containing a gene expressing the SV40 T-antigen on its chromosome
may be used to transform with a vector containing the SV40
replication origin (e.g., pcD). Examples of replication origins to
be used in the present invention include those derived from
polyomavirus, adenovirus, bovine papilomavirus (BPV), and such.
Moreover, to amplify the gene copies in host cell lines, the
expression vector may include an aminoglycoside transferase (APH)
gene, thymidine kinase (TK) gene, E. coli xanthine guanine
phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase
(dhfr) gene, and such as a selective marker.
[0083] On the other hand, in vivo expression of a DNA of the
present invention in animals may be performed by, for example, by
inserting a DNA of the present invention into an appropriate vector
and introducing the vector into the body using retrovirus,
liposome, cationic liposome, adenovirus, and so on. It is possible
to use these methods to perform gene therapy for diseases that
arise from mutations in the NR12 gene of the present invention.
Examples of vectors used for this purpose include, for example,
adenovirus vectors (for example pAdexlcw), retrovirus vectors (for
example, pZIPneo), and such, but are not limited thereto. General
gene manipulations, for example, insertion of the DNA of the
present invention into a vector, may be performed by using standard
methods (Molecular Cloning, 5.61-5.63). The vector may be
administered to a living body through ex vivo or in vivo
methods.
[0084] Another object of the present invention is to provide a
transformant that contains a DNA or vector of the present
invention. The host cell to insert a vector of the invention is not
limited in any way, and for example, E. coli, a variety of animal
cells, and so on may be used. The host cells of the present
invention may be, for example, used as a production system for
preparing or expressing a protein of the present invention. In
vitro and in vivo production systems are known as production system
for producing proteins. Production systems using eukaryotic cells
and prokaryotic cells may be used as the in vitro production
systems.
[0085] When using eukaryotic cells, production system using, for
example, animal cells, plant cells, and fungal cells are available
as hosts. Exemplary animal cells to be used include mammalian cells
such as CHO (J. Exp. Med. 108:945, 1995), COS, 3T3, myeloma, baby
hamster kidney (BHK), HeLa, Vero cells; amphibian cells such as
Xenopus oocytes (Valle et al. Nature 291:338-340, 1981); and insect
cells such as Sf9, Sf21, or Tn5. As CHO cells, especially DHFR
gene-deficient CHO cell, dhfr-CHO (Proc. Natl. Acad. Sci. USA
77:4216-4220, 1980), and CHO K-1 (Proc. Natl. Acad. Sci. USA
60:1275, 1968) can be suitably used. For large-scale preparation in
animal cells, CHO cells may be preferably used. The vector may be
introduced into host cells, for example, by the calcium phosphate
method, the DEAE dextran method, methods using cationic liposome
DOTAP (Boehringer Mannheim), the electroporation method, the
lipofection method, and so on.
[0086] Nicotiana tabacum-derived cells are well known as protein
production systems in plant cells, and these can be callus
cultured. As fungal cells, yeasts such as the Saccharomyces genus,
for example, Saccharomyces cerevisiae; filamentous fungi, such as
Aspergillus genus, for example, Aspergillus niger are known.
[0087] Bacterial cells may be used as prokaryotic production
system. As bacterial cells, E. coli, for example, JM109,
DH5.alpha., HB101, and such, as well as others like Bacillus
subtilis are known.
[0088] Proteins can be obtained by transforming these cells with
the objective DNA, and culturing the transformed cells in vitro.
Transformants can be cultured according to known methods. For
example, DMEM, MEM, RPMI1640, and IMDM can be used as culture media
of animal cells. Occasionally, fetal calf serum (FCS) and such
serum supplements may be added in the above media; alternatively, a
serum-free culture medium may be used. The pH of the culture medium
is preferably from about pH 6 to 8. The culturing is usually
performed at about 30 to 40.degree. C., for about 15 to 200 hr, and
the culture medium changes, aeration, and stirring are done as
necessary.
[0089] On the other hand, for example, production systems using
animals and plants may be given as in vivo protein production
systems. The objective DNA is introduced into the animal or plant,
and the protein is produced within the plant or animal, and then,
the protein is recovered. The term "host" as used in the present
invention encompasses such animals and plants as well.
[0090] When using animals, mammalian and insect production systems
can be used. As mammals, goats, pigs, sheep, mice, and bovines may
be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993).
Alternatively, transgenic animals may also be used when using
mammals.
[0091] For instance, the objective DNA may be prepared as a fusion
gene with a gene encoding a protein intrinsically produced into
milk, such as goat .beta. casein. Next, the DNA fragment comprising
the fusion gene is injected into goat's embryo, and this embryo is
implanted in female goat. The objective protein can be recovered
from the milk of the transgenic goats produced from the goat that
received the embryo and offspring thereof. To increase the amount
of protein-containing milk produced from the transgenic goat, a
suitable hormone(s) may be administered to the transgenic goats
(Ebert et al., Bio/Technology 12:699-702, 1994).
[0092] Silk worms may be used as insects. When using silk worms,
they are infected with baculoviruses to which the DNA encoding
objective protein has been inserted, and the desired protein can be
recovered from body fluids of the silk worm (Susumu et al., Nature
315:592-594, 1985).
[0093] When using plants, for example, tobacco can be used. In the
case of tobacco, the DNA encoding the objective protein is inserted
into a plant expression vector, for example, pMON530, and this is
inserted into a bacteria, such as Agrobacterium tumefaciens. This
bacterium is infected to tobacco, for example, Nicotiana tabacum,
and it is able to obtain the desired polypeptide from the tobacco
leaves (Julian et al., Eur. J. Immunol. 24:131-138, 1994).
[0094] Thus-obtained proteins of the present invention are isolated
from inside or outside (e.g., medium) of the host cell, and may be
purified as a substantially pure homogeneous protein. The
separation and purification of the protein can be done using
conventional separation and purification methods used to purify
proteins and are not limited to any specific method. For instance,
column chromatography, filter, ultrafiltration, salt precipitation,
solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric point electrophoresis, dialysis, recrystallization, and
such may be suitably selected, or combined to separate and purify
the protein.
[0095] For example, affinity chromatography, ion-exchange
chromatography, hydrophobic chromatography, gel filtration, reverse
phase chromatography, adsorption chromatography, and such can be
exemplified as chromatographies (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 performed by liquid
chromatography, such as HPLC, FPLC, and such. The present invention
encompasses proteins highly purified by using such purification
methods.
[0096] Proteins can be arbitrarily modified, or peptides can be
partially excised by treating the proteins with appropriate protein
modification enzymes prior to or after the purification. Trypsin,
chymotrypsin, lysyl-endopeptidase, protein kinase, glucosidase, and
such are used as protein modification enzymes.
[0097] The present invention also provides antibodies that bind to
the protein of the invention. There is no particular restriction as
to the form of the antibody of the invention and the present
invention includes polyclonal antibodies, as well as monoclonal
antibodies. The antiserum obtained by immunizing animals such as
rabbits with a protein of the invention, as well polyclonal and
monoclonal antibodies of all classes, human antibodies, and
humanized antibodies produced by genetic recombination, are also
included.
[0098] A protein of the invention that is used as a sensitizing
antigen for obtaining antibodies is not restricted by the animal
species from which it is derived, but is preferably a protein
derived from mammals, for example, humans, mice, or rats,
especially preferably from humans. Protein of human origin can be
obtained by using the nucleotide sequence or amino acid sequences
disclosed herein.
[0099] Herein, an intact protein or its partial peptide may be used
as the antigen for immunization. As partial peptides of the
proteins, for example, the amino (N)-terminal fragment of the
protein, and the carboxy (C)-terminal fragment can be given.
"Antibody" as used herein means an antibody that specifically
reacts with the full-length or fragments of the protein.
[0100] A gene encoding a protein of the invention or a fragment
thereof is inserted into a known expression vector, and, by
transforming the host cells with the vector described herein, the
desired protein or a fragment thereof is recovered from outside or
inside the host cells using standard methods. This protein can be
used as the sensitizing antigen. Also, cells expressing the
protein, cell lysates, or a chemically synthesized protein of the
invention may be also used as a sensitizing antigen.
[0101] The mammal that is immunized by the sensitizing antigen is
not restricted; however, it is preferable to select animals by
considering the compatibility with the parent cells used in cell
fusion. Generally, animals belonging to the rodentia, lagomorpha,
and Primates are used.
[0102] Examples of animals belonging to rodentia that may be used
include, for example, mice, rats, hamsters, and such. Examples of
animals belonging to lagomorpha that may be used include, for
example, rabbits. Examples of animals of Primates that may be used
include, for example, monkeys. Examples of monkeys to be used
include the infraorder catarrhini (old world monkeys), for example,
Macaca fascicularis, rhesus monkeys, sacred baboons, chimpanzees,
and such.
[0103] Well-known methods may be used to immunize animals with the
sensitizing antigen. For example, the sensitizing antigen is
injected intraperitoneally or subcutaneously into mammals.
Specifically, the sensitizing antigen is suitably diluted and
suspended in physiological saline, phosphate-buffered saline (PBS),
and so on, and mixed with a suitable amount of general adjuvant if
desired, for example, with Freund's complete adjuvant. Then, the
solution is emulsified and injected into the mammal. Thereafter,
the sensitizing antigen suitably mixed with Freund's incomplete
adjuvant is preferably given several times every 4 to 21 days. A
suitable carrier can also be used when immunizing and animal with
the sensitizing antigen. After the immunization, the elevation in
the level of serum antibody is detected by usual methods.
[0104] Polyclonal antibodies against the proteins of the present
invention can be prepared as follows. After verifying that the
desired serum antibody level has been reached, blood is withdrawn
from the mammal sensitized with antigen. Serum is isolated from
this blood using conventional methods. The serum containing the
polyclonal antibody may be used as the polyclonal antibody, or
according to needs, the polyclonal antibody-containing fraction may
be further isolated from the serum. For instance, a fraction of
antibodies that specifically recognize the protein of the invention
may be prepared by using an affinity column to which the protein is
coupled. Then, the fraction may be further purified by using a
Protein A or Protein G column in order to prepare immunoglobulin G
or M.
[0105] To obtain monoclonal antibodies, after verifying that the
desired serum antibody level has been reached in the mammal
sensitized with the above-described antigen, immunocytes are taken
from the mammal and used for cell fusion. For this purpose,
splenocytes can be mentioned as preferable immunocytes. As parent
cells fused with the above immunocytes, mammalian myeloma cells are
preferably used. More preferably, myeloma cells that have acquired
the feature, which can be used to distinguish fusion cells by
agents, are used as the parent cell.
[0106] The cell fusion between the above immunocytes and myeloma
cells can be conducted according to known methods, for example, the
method by Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46,
1981).
[0107] The hybridoma obtained from cell fusion is selected by
culturing the cells in a standard selection medium, for example,
HAT culture medium (medium containing hypoxanthine, aminopterin,
and thymidine). The culture in this HAT medium is continued for a
period sufficient enough for cells (non-fusion cells) other than
the objective hybridoma to perish, usually from a few days to a few
weeks. Then, the usual limiting dilution method is carried out, and
the hybridoma producing the objective antibody is screened and
cloned.
[0108] Other than the above method for obtaining hybridomas, by
immunizing an animal other than humans with the antigen, a
hybridoma producing the objective human antibodies having the
activity to bind to proteins can be obtained by the method of
sensitizing human lymphocytes, for example, human lymphocytes
infected with the EB virus, with proteins, protein-expressing
cells, or lysates thereof in vitro and fusing the sensitized
lymphocytes with myeloma cells derived from human, for example,
U266, having a permanent cell division ability (Unexamined
Published Japanese Patent Application (JP-A) No. Sho 63-17688).
[0109] The monoclonal antibodies obtained by transplanting the
obtained hybridomas into the abdominal cavity of a mouse and
extracting ascites can be purified by, for example, ammonium
sulfate precipitation, protein A or protein G column, DEAE ion
exchange chromatography, an affinity column to which the protein of
the present invention is coupled, and so on. An antibody of the
present invention may be used for the purification or detection of
a protein of the present invention. It may also be a candidate as
an agonist or antagonist of a protein of the present invention.
Furthermore, it is possible to use it in antibody treatment for
diseases in which the protein is implicated. For the administration
to human body (antibody treatment), human antibodies or humanized
antibodies are preferably used because of their reduced
immunogenicity.
[0110] For example, a human antibody against a protein can be
obtained using hybridomas made by fusing myeloma cells with
antibody-producing cells obtained by immunizing a transgenic animal
comprising a repertoire of human antibody genes with an antigen
such as protein, protein-expressing cells, or lysates thereof (see
WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, and
WO96-34096).
[0111] Other than producing antibodies using hybridoma, antibody
producing immunocytes, such as sensitized lymphocytes that are
immortalized by oncogenes, may also be used.
[0112] Such monoclonal antibodies can be also obtained as
recombinant antibodies produced by using the genetic engineering
technique (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 antibodies
are produced by cloning the encoding DNA from immunocytes, such as
hybridoma or antibody-producing sensitized lymphocytes,
incorporating into a suitable vector, and introducing this vector
into a host to produce the antibody. The present invention
encompasses such recombinant antibodies as well.
[0113] Moreover, the antibody of the present invention may be an
antibody fragment or modified-antibody, so long as it binds to a
protein of the invention. For instance, Fab, F (ab').sub.2, Fv, or
single chain Fv (scFv) in which the H chain Fv and the L chain Fv
are suitably linked by a linker (Huston et al., Proc. Natl. Acad.
Sci. USA 85:5879-5883, 1988) can be given as antibody fragments.
Specifically, antibody fragments are generated by treating
antibodies with enzymes, for example, papain or pepsin.
Alternatively, they may be generated by constructing a gene
encoding an antibody fragment, introducing this into an expression
vector, and expressing this vector in suitable host cells (see, for
example, Co et al., J. Immunol. 152:2968-2976, 1994; Better et al.,
Methods Enzymol. 178:476-496, 1989; Pluckthun et al., Methods
Enzymol. 178:497-515, 1989; Lamoyi, Methods Enzymol. 121:652-663,
1986; Rousseaux et al., Methods Enzymol. 121:663-669, 1986; Bird et
al., Trends Biotechnol. 9:132-137, 1991).
[0114] As modified antibodies, antibodies bound to various
molecules, such as polyethylene glycol (PEG), can be used. The
antibodies of the present invention encompass such modified
antibodies as well. To obtain such a modified antibody, chemical
modifications are done to the obtained antibody. These methods are
already established and conventional in the field.
[0115] An antibody of the present invention may be obtained as a
chimeric antibody, comprising non-human antibody-derived variable
region and human antibody-derived constant region, or as a
humanized antibody comprising non-human antibody-derived
complementarily determining region (CDR), human antibody-derived
framework region (FR), and human antibody-derived constant region
by using conventional methods.
[0116] Antibodies thus obtained can be purified to uniformity. The
separation and purification methods used in the present invention
for separating and purifying the antibody may be any method usually
used for proteins. For example, column chromatography, such as
affinity chromatography, filter, ultrafiltration, salting-out,
dialysis, SDS polyacrylamide gel electrophoresis, isoelectric
focusing, and others, may be appropriately selected and combined to
isolate and purify the antibodies (Antibodies: A Laboratory Manual.
Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988);
however, the invention is not limited thereto. Antibody
concentration of the above mentioned antibody can be assayed by
measuring the absorbance, or by the enzyme-linked immunosorbent
assay (ELISA), etc.
[0117] Protein A or Protein G column can be used for the affinity
chromatography. Protein A column may be, for example, Hyper D,
POROS, Sepharose F. F. (Pharmacia), etc.
[0118] Other chromatography may also be used, for example, such as
ion-exchange chromatography, hydrophobic chromatography, gel
filtration, reverse-phase chromatography, and adsorption
chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These may be
performed on liquid-phase chromatography such as HPLC, FPLC, and so
on.
[0119] Examples of methods that assay the antigen-binding activity
of the antibodies of the invention include, for example,
measurement of absorbance, enzyme-linked immunosorbent assay
(ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or
immunofluorescence. For example, when using ELISA, a protein of the
invention is added to a plate coated with the antibodies of the
present invention, and then, the objective antibody sample, for
example, culture supernatants of antibody-producing cells, or
purified antibodies are added. Then, secondary antibody recognizing
the primary antibody, which is labeled by alkaline phosphatase and
such enzymes, is added, the plate is incubated and washed, and the
absorbance is measured to evaluate the antigen-binding activity
after adding an enzyme substrate such as p-nitrophenyl phosphate.
As the protein, a protein fragment, for example, a fragment
comprising a C-terminus, or a fragment comprising an N-terminus may
be used. To evaluate the activity of the antibody of the invention,
BIAcore (Pharmacia) may be used.
[0120] By using these methods, the antibody of the invention and a
sample presumed to contain a protein of the invention are
contacted, and the protein of the invention is detected or assayed
by detecting or assaying the immune complex formed between the
above-mentioned antibody and the protein.
[0121] A method of detecting or assaying a protein of the invention
is useful in various experiments using proteins as it can
specifically detect or assay the proteins.
[0122] Another object of this invention is to provide a
polynucleotide of at least 15 nucleotides that is complementary to
the DNA encoding human NR12 protein (SEQ ID NO:1, 3, 5, 7 or 9) or
its complementary strand.
[0123] Herein, the term "complementary strand" is defined as one
strand of a double strand polynucleotide composed of A:T and G:C
base pairs to the other strand. Also, "complementary" is defined as
not only those completely matching within a continuous region of at
least 15 nucleotides, but also having a homology of at least 70%,
preferably 80%, more preferably 90%, and most preferably 95% or
higher within that region. The homology may be determined using the
algorithm described herein.
[0124] Probes and primers for detection or amplification of the DNA
encoding a protein of the invention, or a nucleotide or nucleotide
derivative for the suppression of the protein expression (such as,
antisense oligonucleotide and ribozyme) are included in these
polynucleotides. Such polynucleotides may be also used for
preparing DNA chips.
[0125] The antisense oligonucleotides that hybridize with a portion
of the nucleotide sequence of any of SEQ ID NO:1, 3, 5, 7, and 9
are also included in the antisense oligonucleotides of the present
invention. These antisense oligonucleotides are preferably directed
against a sequence which contains at least 15 continuous
nucleotides comprised in any one of the nucleotide sequence of SEQ
ID NO:1, 3, 5, 7, and 9. More preferably, it is the antisense
oligonucleotide against at least 15 continuous nucleotides
containing a translation start codon.
[0126] Derivatives or modified products of antisense
oligonucleotides can be used as antisense oligonucleotides.
Examples of such modified products include, for example, lower
alkyl phosphonate modifications such as methyl-phosphonate-type or
ethyl-phosphonate-type; phosphorothioate modifications;
phosphoroamidate modifications, and such.
[0127] The term "antisense oligonucleotides" as used herein means,
not only those in which the nucleotides corresponding to those
constituting a specified region of a DNA or mRNA are entirely
complementary, but also those having a mismatch of one or more
nucleotides, so long as the DNA or mRNA and the oligonucleotide can
specifically hybridize with the nucleotide sequence of SEQ ID NO:1,
3, 5, 7 or 9.
[0128] The antisense oligonucleotide derivatives of the present
invention act upon cells producing a protein of the invention by
binding to the DNA or RNA encoding the protein to inhibit its
transcription or translation, and to promote the degradation of the
mRNA, and have an effect of suppressing the function of the protein
of the invention by suppressing the expression of the protein.
[0129] An antisense oligonucleotide derivative of the present
invention can be made into an external preparation, such as a
liniment or a poultice, by mixing with a suitable base material,
which is inactive against the derivatives.
[0130] Also, as needed, the derivatives can be formulated into
tablets, powders, granules, capsules, liposome capsules,
injections, solutions, nose-drops, freeze-dried agents, and such by
adding excipients, isotonic agents, solubilizing agents,
stabilizers, preservative substance, pain-killers, and such. These
can be prepared using conventional methods.
[0131] An antisense oligonucleotide derivative is given to the
patient by directly applying it onto the ailing site, by injecting
it into the blood vessel and such, so that it will reach the ailing
site. An antisense-mounting medium can also be used to increase
durability and membrane-permeability. Examples are, liposome,
poly-L-lysine, lipid, cholesterol, lipofectin or derivatives of
them.
[0132] The dosage of the antisense oligonucleotide derivative of
the present invention can be adjusted suitably according to the
patient's condition and used in desired amounts. For example, a
dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be
administered.
[0133] The antisense oligonucleotide of the present invention is
useful in inhibiting the expression of the protein of the
invention, and therefore is useful in suppressing the biological
activity of the proteins of the invention. Also,
expression-inhibitors comprising the antisense oligonucleotide of
the invention are useful, because of their capability to suppress
the biological activity of the proteins of the invention.
[0134] Proteins of this invention are useful in screening for
compounds that bind to the protein. That is, the proteins are used
in a method of screening for compounds that bind to the proteins of
this invention, in which the method comprises bringing proteins of
this invention into contact with a test sample that is expected to
contain a compound that may bind to the proteins and selecting the
compound with the activity of binding to the proteins of the
invention.
[0135] Proteins of this invention to be used in the screening of
the invention may be any of recombinant, natural, or partial
peptides. Also, they may be in the form of proteins expressed on
the cell surface or membrane fractions. Test samples to be used in
the screening method of the present invention are not limited, but
may be, for example, cell extracts, cell culture supernatants,
microbial fermentation products, extracts of marine organisms,
plant extracts, purified or partly purified proteins, peptides,
non-peptide compounds, synthetic low molecular compounds, or
natural compounds. The proteins of this invention may be exposed to
the sample as purified protein or soluble proteins, in a form bound
to a carrier, as fusion proteins with another protein, in a form
expressed on the cell surface, or as membrane fractions.
[0136] A protein of the present invention may be used to screen for
other proteins that bind to the target protein (such as ligands)
using a variety of methods known to one skilled in the art. These
screening processes can be carried out, for example, by the
immunoprecipitation method. Specifically, the method can be carried
out as follows. The gene encoding a protein of the present
invention is expressed by inserting the gene into an expression
vector for foreign gene expression like pSV2neo, pcDNA I, pCD8, and
such, and expressing the gene in animal cells, etc. Any generally
used promoters may be employed for the expression, including the
SV40 early promoter (Rigby in Williamson (ed.), Genetic
engineering, Vol. 3. Academic Press, London, p. 83-141, 1982), the
EF-1.alpha. promoter (Kim et al., Gene 91:217-223, 1990), the CAG
promoter (Niwa et al., Gene 108:193-200, 1991), the RSV LTR
promoter (Cullen, Methods in Enzymology 152:684-704, 1987), the
SRo: promoter (Takebe et al., Mol. Cell. Biol. 8:466, 1988), the
CMV immediate early promoter (Seed et al., Proc. Natl. Acad. Sci.
USA 84:3365-3369, 1987), the SV40 late promoter (Gheysen et al., J.
Mol. Appl. Genet. 1:385-394, 1982), the Adenovirus late promoter
(Kaufman et al., Mol. Cell. Biol. 9:946, 1989), the HSV TK
promoter, and soon.
[0137] Transfer of a foreign gene into animal cells for its
expression can be performed by any of the following methods,
including the electroporation method (Chu et al., Nucl. Acid Res.
15:1311-1326, 1987), the calcium phosphate method (Chen et al.,
Mol. Cell. Biol. 7:2745-2752, 1987), the DEAE dextran method
(Lopata et al., Nucl. Acids Res. 12:5707-5717, 1984; Sussman et
al., Mol. Cell. Biol. 4:1642-1643, 1985), the lipofectin method
(Derijard, Cell 7:1025-1037, 1994; Lamb et al., Nature Genetics
5:22-30, 1993; Rabindran et al., Science 259:230-234, 1993), and
such. A protein of the present invention can be expressed as a
fusion protein having the recognition site (epitope) for a
monoclonal antibody by introducing a recognition site (epitope) for
a monoclonal antibody, the specificity of which has been
established, into the N- or C-terminus of the protein of the
present invention. For this purpose, a commercial epitope-antibody
system can be utilized (Exp. Med. 13:85-90, 1995). Vectors are
commercially available which are capable of expressing fusion
proteins with .beta.-galactosidase, maltose binding protein,
glutathione S-transferase, green florescence protein (GFP), and
such, via the multi-cloning site.
[0138] To minimize the alteration of the properties of a protein of
this invention arising from the formation into a fusion protein, a
the fusion protein may be prepared by introducing only a small
epitope portion comprising several to ten amino acids as reported
in the literature. For example, the epitopes of polyhistidine
(His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular
stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein
(T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag
(epitope on the monoclonal phage), and such, and monoclonal
antibodies to recognize these epitopes can be utilized as the
epitope-antibody system for screening proteins binding to the
protein of the present invention (Exp. Med. 13:85-90, 1995).
[0139] In immunoprecipitation, immune complexes are formed by
adding these antibodies to cell lysate prepared using suitable
detergents. This immune complex consists of a protein of the
present invention, a protein capable of binding to the protein, and
an antibody. The immunoprecipitation can also be conducted using an
antibody against a protein of the present invention besides
antibodies against the above epitopes. An antibody against the
protein of the present invention can be prepared, for example, by
inserting a gene encoding a protein of the present invention into
an appropriate E. coli expression vector to express the gene in E.
coli, purifying the expressed protein, and immunizing rabbits,
mice, rats, goats, chickens, and such, with the purified protein.
The antibody can also be prepared by immunizing the above-described
animals with partial peptides of the protein of the present
invention.
[0140] Immune complexes can be precipitated using, for example,
Protein A Sepharose or Protein G Sepharose in case where the
antibody is a mouse IgG antibody. In addition, in the case where
the protein of the present invention is prepared as a fusion
protein with the epitope of, for example, GST, and such, the immune
complex can be formed using a substance that specifically binds to
these epitopes, such as glutathione-Sepharose 4B, and such, giving
the same result as in the case where the antibody for the protein
of the present invention is used.
[0141] Immunoprecipitation, in general, may be performed following,
or according to, for example, the method described in the
literature (Harlow, E. and Lane, D.: Antibodies pp. 511-552, Cold
Spring Harbor Laboratory publications, New York, 1988).
[0142] SDS-PAGE is generally used for the analysis of
immunoprecipitated proteins, and bound proteins can be analyzed
based on the molecular weight of proteins using a gel of an
appropriate concentration. In this case, although proteins bound to
a protein of the present invention, in general, are hardly
detectable by the usual protein staining methods, such as Coomassie
staining and silver staining, the detection sensitivity can be
improved by culturing cells in a culture medium containing radio
isotope-labeled .sup.35S-methionine and .sup.35S-cystein to label
proteins inside the cells, and detecting the labeled proteins. Once
the molecular weight of the protein is determined, the desired
protein can be purified directly from SDS-polyacrylamide gel and
sequenced.
[0143] In addition, isolation of proteins binding to a protein of
the present invention can be also performed using, for example, the
West-Western blotting analysis (Skolnik et al., Cell 65:83-90,
1991). Specifically, a cDNA library is constructed from cells,
tissues, and organs in which protein binding to the protein of this
invention is expected to be expressed by using phage vectors (such
as, .lamda.gt11 and ZAP), proteins expressed on LB-agarose are
fixed on a filter, which is then reacted with a purified and
labeled protein of the present invention, and plaques expressing
proteins binding to the protein of the present invention are
detected by the label. Methods for labeling a protein of the
invention include methods utilizing the binding of biotin and
avidin, methods utilizing antibodies specifically binding to the
protein of the present invention, or peptides or polypeptides (for
example, GST, etc.) fused with the protein of the present
invention, methods utilizing radioisotope and fluorescence, and
such.
[0144] Further, another embodiment of the screening method of the
present invention is exemplified by a method utilizing the
two-hybrid system using cells (Fields et al., Trends Genet.
10:286-292, 1994; Dalton et al., Cell 68:597-612, 1992; "MATCHMAKER
Two-Hybrid System", "Mammalian MATCHMAKER Two-Hybrid Assay Kit",
"MATCHMAKER One-Hybrid System" (all from Clontech); "HybriZAP
Two-Hybrid Vector System" (Stratagene)). In the two-hybrid system,
a protein of this invention may be fused to the DNA binding domain
of SRF or GAL4, and expressed in yeast cells. A cDNA library is
constructed from cells predicted to express proteins that bind to
the protein of this invention, wherein the cDNA library is
constructed in such a way that the proteins are expressed as fusion
proteins with transcription activation regions of VP16 or GAL4. The
cDNA library is transfected into the aforementioned yeast cells,
and then positive clones are detected so as to isolate the
cDNA-derived from the library (i.e., expression of a protein that
binds to the protein of the invention in yeast cell leads to the
binding of the two proteins, and results in the activation of the
reporter gene, which allows for the detection positive clones). The
protein encoded by the isolated cDNA may be obtained by introducing
the cDNA into E. coli and expressing it therein. Thus, it is
possible to prepare proteins that bind to a protein of this
invention and genes encoding them. The reporter gene to be used in
the two-hybrid system may be any suitable gene, such as HIS3 gene,
Ade2 gene, LacZ gene, CAT gene, luciferase gene, or plasminogen
activator inhibitor type1 (PAI-1) gene, but is not limited
thereto.
[0145] Screening for compounds which bind to a protein of the
present invention can be also carried out using affinity
chromatography. For example, a protein of the invention is
immobilized on a carrier in the affinity chromatography column, to
which a test sample, which is expected to express a protein binding
to the protein of the invention, is applied. Samples may be, for
example, cell extracts, cell lysates, etc. After loading the test
sample, the column is washed, and proteins which binds to the
protein of the invention can be obtained.
[0146] The obtained protein may be analyzed for its amino acid
sequence to synthesize oligonucleotide probes, which may then be
used to screen a cDNA library to obtain a DNA encoding the
protein.
[0147] A biosensor that utilizes surface plasmon resonance
phenomenon may be used to detect or measure the bound compound.
Such biosensors (for example, BIAcore (Pharmacia)) may enable the
observation of the interaction at real-time using a small amount of
protein without the need for labeling. Thus, it is possible to
assess the interaction between the protein of the invention and
test compounds using biosensors such as BIAcore.
[0148] Moreover, compounds that bind to a protein of the invention
(including agonists and antagonists), which compounds are not
always proteins, may be isolated using a variety of methods known
to one skilled in the art. For instance, the protein of the
invention may be fixed and exposed to synthetic compounds, a bank
of natural substances, or a random phage peptide display library to
screen for molecules that bind to the protein. Alternatively,
high-throughput screening using combinatorial chemistry techniques
may be performed (Wrighton et al., Science 273:458-64, 1996;
Verdine, Nature 384:11-13, 1996; Hogan, Jr., Nature 384:17-9,
1996).
[0149] Screening of a ligand that binds to a protein of the
invention may be performed as follows. The extracellular domain of
a protein of the invention is fused to the intracellular domain
including the transmembrane domain of a hemopoietin receptor
protein that has a known signal transducing ability to prepare a
chimeric receptor. The chimeric receptor may be expressed on the
cell surface of a suitable cell line, preferably a cell line that
can survive and proliferate only in the presence of a suitable
growth proliferative factor (growth factor-dependent cell line).
Then, the cell line may be cultured in medium supplemented with a
sample material in which a variety of growth factors, cytokines, or
hemopoietic factors might be expressed. According to this method,
the growth factor-dependent cell line can only survive and
proliferate when the sample contains an appropriate ligand that
specifically binds to the extracellular domain of the protein of
the invention. The known hemopoietin receptors, such as the
thrombopoietin receptor, erythropoietin receptor, G-CSF receptor,
gp 130, and so on may be used. The partner for constructing a
chimeric receptor for the screening system of the invention is not
limited to the above receptors so long as its intracellular domain
provides a structure necessary for the signal transduction
activity. The growth factor-dependent cell line may be, for
example, IL-3-dependent cell lines such as BaF3 or FDC-P1.
[0150] In a rare case, the ligand that specifically binds to a
protein of the invention may not be a soluble protein but a
membrane-bound protein. In this case, screening can be done using a
protein comprising only the extracellular domain of the protein of
the invention, or a fusion protein in which the extracellular
domain is attached to a part of other soluble proteins. Such
proteins are labeled before they are used for measuring the binding
with the cells that are expected to express the ligand. The former
protein, comprising only the extracellular domain, may be a soluble
receptor protein artificially constructed through introducing a
stop codon into the N-terminal side of the transmembrane domain, or
a soluble protein such as NR12-1. The latter fusion protein may be
a protein in which the Fc region of immunoglobulin, or FLAG peptide
is attached to the C-terminus of the extracellular domain. These
labeled soluble proteins can also be useful in detection by the
above-described West-western blotting method.
[0151] A chimeric protein of the extracellular domain of a protein
of this invention and the Fc region of an antibody (such as human
IgG antibody) may be purified using Protein A column, etc. Such an
antibody-like chimeric protein retains its ligand binding activity.
Thus, the protein may be appropriately labeled with an isotope and
so on, and used for the screening of a ligand (Suda et al., Cell
175:1169-1178, 1993). Some cytokines, such as molecules of the TNF
family, primarily exist in a membrane-bound form, so such ligands
may be isolated by exposing the antibody-like chimeric protein to a
variety of cells and selecting cells based on the binding activity
to the protein. Alternatively, ligands may be isolated according to
the same method by using cells to which a cDNA library is
introduced. Furthermore, the antibody-like chimeric protein may
also be used as an antagonist.
[0152] The compound isolated by the above screening may be a
candidate for drugs that activate or inhibit the activity of a
protein of this invention. It is possible to apply such compounds
for the treatment of the disease arising from aberrant expression
or functional disorder of a protein of the present invention. The
compound obtained using the screening method of the invention
includes compounds resulting from the modification of the compound
having the activity to bind to the protein of the invention by
adding, deleting, and/or replacing a part of the structure.
[0153] When using the isolated compound or a protein of the present
invention (decoy type (soluble form)) as a pharmaceutical for
humans and other mammals, for example, mice, rats, guinea pigs,
rabbits, chicken, cats, dogs, sheep, pigs, bovines, monkeys, sacred
baboons, chimpanzees, the isolated compound can be directly
administered or can be formulated into a dosage form using known
pharmaceutical preparation methods. For example, according to the
need, the pharmaceuticals can be taken orally, as sugar-coated
tablets, capsules, elixirs and microcapsules, or parenterally, in
the form of injections of sterile solutions, suspensions with
water, or any other pharmaceutically acceptable liquid. For
example, the compounds can be mixed with a pharmacologically
acceptable carrier or medium, specifically, sterilized water,
physiological saline, plant-oil, emulsifiers, suspending agent,
surfactants, stabilizers, flavoring agents, excipients, vehicles,
preservatives and binders, in a unit dosage form required for
generally accepted drug implementation. The amount of active
ingredient in these preparations makes a suitable dosage acquirable
within the indicated range.
[0154] Examples of additives which can be mixed to tablets and
capsules include: binders, such as gelatin, corn starch, tragacanth
gum and gum acacia; excipients, such as crystalline cellulose;
swelling agents, such as corn starch, gelatin and alginic acid;
lubricants, such as magnesium stearate; and sweeteners, such as
sucrose, lactose or saccharin; flavoring agents, such as
peppermint, Gaultheria adenothrix oil and cherry. When the unit
dosage form is a capsule, a liquid carrier, such as oil, can also
be included in the above ingredients. Sterile composites for
injections can be formulated following normal drug implementations,
using vehicles such as distilled water used for injections.
[0155] For example, physiological saline, glucose, and other
isotonic liquids, including adjuvants, such as D-sorbitol,
D-mannose, D-mannitol, and sodium chloride, can be used as aqueous
solutions for injections. These can be used in conjunction with
suitable solubilizers, such as alcohol, specifically ethanol,
polyalcohols such as propylene glycol and polyethylene glycol, and
non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
[0156] Sesame oil or soy-bean oil can be used as an oleaginous
liquid and may be used in conjunction with benzyl benzoate or
benzyl alcohol as solubilizers; may be formulated with a buffer
such as phosphate buffer and sodium acetate buffer; and may be used
in conjunction with a pain-killer such as procaine hydrochloride, a
stabilizer such as benzyl alcohol and phenol, and an anti-oxidant.
The prepared injection is filled into a suitable ampule.
[0157] Methods well known to those skilled in the art may be used
to administer the pharmaceutical compound to patients, for example
as intraarterial, intravenous, percutaneous injections and also as
intranasal, transbronchial, intramuscular or oral administrations.
The dosage varies according to the body-weight and age of the
patient, the administration method, and such, but one skilled in
the art can suitably select them. If said compound is encodable by
a DNA, said DNA can be inserted into a vector for gene therapy to
perform the therapy. The dosage and method of administration vary
according to the body-weight, age, and symptoms of a patient, but
one skilled in the art can select them suitably.
[0158] For example, the dosage of the protein of this invention
(decoy form (soluble form)) may vary depending on the subject of
administration, target organ, symptom, and method for
administration. However, it may be injected to a normal adult (body
weight, 60 kg) at a dose of about 100 .mu.g to 10-20 mg per
day.
[0159] For example, although there are some differences according
to the symptoms, the dose of a compound that binds with a protein
of the present invention, or a compound that inhibits the activity
of a protein of this invention is typically about 0.1 mg to about
100 mg per day, preferably about 1.0 mg to about 50 mg per day, and
more preferably about 1.0 mg to about 20 mg per day, when
administered orally to a normal adult (weight 60 kg).
[0160] When the protein is administered parenterally, in the form
of an injection to a normal adult (weight 60 kg), although there
are some differences according to the patient, target organ,
symptoms and method of administration, it is convenient to
intravenously inject a dose of about 0.01 mg to about 30 mg per
day, preferably about 0.1 to about 20 mg per day and more
preferably about 0.1 to about 10 mg per day. Also, in the case of
other animals, it is possible to administer an amount converted to
60 kg of body-weight or surface area.
[0161] All publications and patents cited herein are incorporated
by reference in their entirety.
DESCRIPTION OF DRAWINGS
[0162] FIG. 1 shows the partial nucleotide sequence of AL109843
identified in the htgs database (SEQ ID NO:23). The deduced amino
acid sequence is shown under the predicted exon sequence (SEQ ID
NO:24). The YR motif sequence and WS motif that were used as the
target are boxed.
[0163] FIG. 2 shows partial amino acid sequences of NR12 found in
the sequence of AL109843 (from the top, SEQ ID NOs:25-29), and
those of known hemopoietin receptors having homology thereto.
Identical amino acid sequences are boxed and similar amino acid
sequences are shadowed. Gap spaces are underlined. Known
hemopoietin receptors are, from top, human gp130 (SEQ ID NO:30),
human NR9 (SEQ ID NO:31), human prolactin receptor (SEQ ID NO:32),
human IL-7 receptor (SEQ ID NO:33), and human LIF receptor (SEQ ID
NO:34).
[0164] FIG. 3 shows a photograph demonstrating the results of PCR
analysis, showing expressed products amplified by 5'-RACE and
3'-RACE using the oligonucleotide primers designed against the
predicted WS exon within the AL109843 sequence. Specific products
by PCR are shown with arrows.
[0165] FIG. 4 shows the nucleotide sequence of the full-length
NR12.1 cDNA that was obtained by combining the 5'-RACE and 3'-RACE
products (SEQ ID NO:1). The deduced amino acid sequence encoded by
NR12.1 is also shown (SEQ ID NO:2). The amino acid sequence
predicted to be the secretion signal is underlined. Conserved
cysteine residues, and the amino acid sequences of YR motif and WS
motif are boxed.
[0166] FIG. 5 shows the nucleotide sequence of the full-length
NR12.2 cDNA that was obtained by combining the 5'-RACE and 3'-RACE
products (SEQ ID NO:3). The amino acid sequence encoded by NR12.2
is also shown (SEQ ID NO:4). The predicted secretion signal
sequence is underlined. The predicted transmembrane region is
shadowed. Conserved cysteine residues in the extracellular region,
and amino acid sequences of YR motif and WS motif are boxed.
[0167] FIG. 6 shows the nucleotide sequence of full-length NR12.3
cDNA that was obtained by combining the 5'-RACE and 3'-RACE
products (SEQ ID NO:5). The amino acid sequence encoded by NR12.3
is also shown (SEQ ID NO:6). The predicted secretion signal is
underlined. Conserved cysteine residues, and the amino acid
sequences of YR motif and WS motif are boxed.
[0168] FIG. 7 is a continuation of FIG. 6.
[0169] FIG. 8 shows photographs demonstrating the results of RT-PCR
analysis of the genetic-expression distribution of the NR12 in
human organs. The arrow indicates the size of the specific PCR
amplification product of NR12.
[0170] FIG. 9 shows a photograph demonstrating the results of
quantification of the NR12 gene expression in human organs by
Southern blotting. The arrow indicates the size of the specific
signal of detected NR12.
[0171] FIG. 10 is a schematic illustration of the structure of the
NR12 fusion protein to be expressed from the expression vector
construct in the mammalian cell.
[0172] FIG. 11 shows the nucleotide sequence of full-length NR12.4
cDNA that was obtained by combining the 5'-RACE and 3'-RACE
products (SEQ ID NO:7). The amino acid sequence encoded by NR12.4
is also shown (SEQ ID NO:8). The predicted secretion signal is
underlined. Conserved cysteine residue, and amino acid sequences of
YR motif and WS motif are boxed.
[0173] FIG. 12 is a continuation of FIG. 11.
[0174] FIG. 13 shows the nucleotide sequence of full-length NR12.5
cDNA (SEQ ID NO:9). The amino acid sequence encoded by NR12.5 is
also shown (SEQ ID NO:10). The predicted secretion signal is
underlined. The predicted transmembrane sequence is shaded.
Conserved cysteine residue in the extracellular region, and amino
acid sequences of YR motif and WS motif are boxed.
[0175] FIG. 14 is a continuation of FIG. 13.
DETAILED DESCRIPTION
[0176] The present invention will be explained below with reference
to examples, but it is not construed as being limited thereto.
Example 1
Isolation of NR12 Gene
(1) Primary Screening by TblastN Search
[0177] Although sequencing of human genome is promoted extensively
by human genome projects of institutes, the proportion of
completely finished sequences to the whole human genome has not
reached even 10%. However, information provided by above projects
until today is counted as a good means for searching target genes,
determining nucleotide sequences, and mapping genes. The
informational basis of the above sequences consists of large
information provided by the assembly of bacterial artificial
chromosome (BAC) and yeast artificial chromosome (YAC), which aims
to form a complete database in the future. The present inventors
identified a human gene encoding a part of a novel hemopoietin
receptor protein from a BAC clone sequence in one of public
databases, "High Throughput Genomic Sequence (htgs)" of
GenBank.
[0178] As mentioned above, the present inventors found motif
sequences conserved in the hemopoietin receptor family, namely
(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg motif (YR
motif) in the extracellular region, and Trp-Ser-Xaa-Trp-Ser (SEQ ID
NO:21) motif (WS motif) located around the C-terminus. However, it
is extremely difficult to design oligonucleotide probe that
includes both motif sequences comprehensively. Therefore, the
inventors conducted in silico database search using partial amino
acid sequences from the fragment of known hemopoietin receptor
proteins including both motifs as the query. Fragmentation of
partial amino acid sequences that may be used as a query was
examined using the human receptors shown in table 1 as the sequence
of known hemopoietin receptors. According to the genomic structure
of the known hemopoietin receptor sequences, the exons encoding
these YR motif and WS motif were about 50 to 70 amino acids long,
and the exon proximal to it to the N-terminus (PP exon) was also
about 50 to 70 amino acids long. Thus, a sequence containing both
exons consisting of about 120 amino acids were cut to prepare a
query sequence for convenience' sake. Although the length of the
partial amino acid sequence used as the query sequence varied
depending on each known hemopoietin receptor, the feature of the
structure was conserved. A sequence that ranges from one or more
proline residues located near the initiation site in the PP exon to
the amino acid residue located about 10 amino acids to the
C-terminus of the WS motif termination in the WS exon was extracted
as the query sequences from all known hematopoietin receptor
sequences.
[0179] The known hemopoietin receptors used as query sequences for
the database search is shown in the table. The amino acid residues
conserved among motif sequences are shown in bold with
underline.
TABLE-US-00001 TABLE 1 Human GenBank YR-motif WS-motif Receptors
Accession # Sequence Sequence LIF-R NM_002310 YTFRIR WSKWS (SEQ ID
NO: 35) (SEQ ID NO: 57) gp130 NM_002184 YVFRIR WSDWS (SEQ ID NO:
36) (SEQ ID NO: 58) IL-12R.beta.1 NP_005526 QEFQLR WSKWS (SEQ ID
NO: 37) (SEQ ID NO: 57) IL-12R.beta.2 NM_001559 YEFQIS WSDWS (SEQ
ID NO: 38) (SEQ ID NO: 58) G-CSFR NM_000760 YTLQIR WSDWS (SEQ ID
NO: 39) (SEQ ID NO: 58) EPO-R M34986 YTFAVR WSAWS (SEQ ID NO: 40)
(SEQ ID NO: 59) TPO-R M90103 YRLQLR WSSWS (SEQ ID NO: 41) (SEQ ID
NO: 60) Leptin-R U50748 YAVQVR WSNWS (SEQ ID NO: 42) (SEQ ID NO:
61) IL-3R.alpha. M74782 YTVQIR LSAWS (SEQ ID NO: 43) (SEQ ID NO:
62) IL-4R NM_000418 YRARVR WSEWS (SEQ ID NO: 44) (SEQ ID NO: 63)
IL-5R.alpha. M96651 YDVQVR WSEWS (SEQ ID NO: 45) (SEQ ID NO: 63)
IL-6R NM_000565 HVVQLR WSEWS (SEQ ID NO: 46) (SEQ ID NO: 63) IL-7R
NM_002185 YEIKVR WSEWS (SEQ ID NO: 47) (SEQ ID NO: 63)
IL-11R.alpha. U32324 HAVRVS WSTWS (SEQ ID NO: 48) (SEQ ID NO: 64)
IL-13R.alpha. NM_001560 NTVRIR WSNWS (SEQ ID NO: 49) (SEQ ID NO:
61) IL-2R.beta. A28052 YEFQVR WSPWS (SEQ ID NO: 50) (SEQ ID NO: 65)
IL-2R.gamma. NM_000206 YTFRVR WSEWS (SEQ ID NO: 51) (SEQ ID NO: 63)
GM-CSFR M64445 HSVKIR WSSWS (SEQ ID NO: 52) (SEQ ID NO: 60) CNTF-R
NM_001842 YIIQVA WSDWS (SEQ ID NO: 53) (SEQ ID NO: 58) PRL-R
NM_000949 YLVQVR WSAWS (SEQ ID NO: 54) (SEQ ID NO: 59) NR6(CRLF1)
NM_004750 YFVQVR WSEWS (SEQ ID NO: 55) (SEQ ID NO: 63) NR9(CREME9)
AF120151 YQFRVC WSPWS (SEQ ID NO: 56) (SEQ ID NO: 65)
[0180] The above queries were used to search on the htgs database
in GenBank using TblastN (Advanced TblastN 2.0.9) program. The
default values (Expect=100, Descriptions=250, and Alignments=250)
were used as parameters for the search. As a result, the search
resulted in many false positive clones, and those clones which both
of the YR motif and WS motif were not encoded in the same reading
frame, or that contained a stop codon between the two motifs were
excluded. Also those clones containing only the YR motif but not
the WS motif were also excluded, because, as mentioned above, the
YR motif is not a completely established consensus sequence.
Therefore, the conservation of the WS motif was considered
predominant. As a result of the above selection, positive clones of
primary search shown in table 2 were chosen from about 1000
pseudo-positive clones obtained by the TblastN search.
[0181] Positive clones obtained by the primary search against htgs
database having the target motif sequence with high probability
were selected, and are shown in the table. Conserved amino acid
residues are shown in bold with underline in the motif
sequences.
TABLE-US-00002 TABLE 2 GenBank Motif Accession # Sequence Note
AC008048 WSPWS IL-2R beta (SEQ ID NO: 65) AC007174 WSEWS IL-5R (SEQ
ID NO: 63) AL031406 WSTWS CH.22 (SEQ ID NO: 64) AC003656 WSGWS
CH.21 (SEQ ID NO: 66) AC008663 WSKWS CH.5 (SEQ ID NO: 57) AC008614
WSGWS CH.5 (SEQ ID NO: 66) AC008532 WSGWS CH.19 (SEQ ID NO: 66)
AC009267 WSTWS CH.18 (SEQ ID NO: 64) AC007596 WSSWS CH.16 (SEQ ID
NO: 60) AC007227 WGEWS CH.16 (SEQ ID NO: 67) AL031123 WSDWA CH.6
(SEQ ID NO: 68) AC005911 WGEWS CH.12 (SEQ ID NO: 67) AL096870 WSNWK
CH.14 (SEQ ID NO: 69) Z97201 WSNWK CH.12 (SEQ ID NO: 69) AC007902
WSGWS CH.18 (SEQ ID NO: 66) AC008536 WSMWS CH.5 (SEQ ID NO: 70)
AC006176 WSGWS CH.10 (SEQ ID NO: 66) AC004846 WSQWS none (SEQ ID
NO: 71) AL109843 WQPWS CH.1 (NR12) (SEQ ID NO: 72) AC003656 WSEWG
CH.21 (SEQ ID NO: 73) AC005143 TSGWS CH.15 (SEQ ID NO: 74) AL109743
WSGWS CH.1 (SEQ ID NO: 66) AC008403 WSAWS CH.19 (SEQ ID NO: 59)
AL032818 WSGWS CH.22 (SEQ ID NO: 66) Z93017 WSGWS CH.6 (SEQ ID NO:
66) AC009456 WSRWS CH.18 (SEQ ID NO: 75) AC008427 WSEGS CH.5 (SEQ
ID NO: 76) AL096791 WSQWS CH.X (SEQ ID NO: 71)
(2) Secondary Screening by BlastX Search
[0182] First, nucleotide sequences around the sequence which were
positive to the query sequence in the primary search were cut from
each of the 28 positive clones of TblastN primary search shown in
table 2. Using these sequences as the query, the nr database in
GenBank was searched again using the BlastX (Advanced BlastX 2.0.9)
program. The query sequence consisted of a nucleotide sequence of
240 bp in total, which contains the sequence approximately 200 bp
upstream of the sequence that may encode the WS motif, for
convenience sake. Because, as mentioned above, the exon encoding
the WS motif was as short as approximately 50 to 70 amino acids in
the genome structure of known hemopoietin receptors, the prepared
query sequence of 240 bp long is expected to cover the exon
sufficiently. The value of "Expect=100, Descriptions=100,
Alignments=100, Filter=default" was used for the BlastX search. It
was expected that positive clones showing at least homology with
multiple different known hemopoietin receptors would be selected as
positive clones of secondary search encoding hematopoietin receptor
family members from the positive clones of the secondary search
according to the search.
[0183] As a result of the above two-step Blast search, three clones
(AC008048, AC007174, and AL109843) among the human genome clones
shown in table 2 were successfully identified as positive clones of
the secondary search. However, AC008048 and AC007174 were revealed
to be genome sequences that encode the human IL-2 receptor beta
strand and human IL-5 receptor, respectively. AL109843 alone was
inferred to encode the target novel hemopoietin receptor.
Therefore, this clone was named NR12, and was determined to isolate
the full-length cDNA.
[0184] AL109843 is a genome draft sequence derived from human
chromosome 1 submitted to htgs database at 16 Aug. 1999, and has a
length of 149104 bp. However, nucleotide sequences at 10 positions,
approximately 8000 bp in total, remains undetermined at this time.
The existence of a WS exon could be predicted in the sequence of
AL109843 which were positive in the TblastN primary search as shown
in FIG. 1. The YR motif, [YVFQVR; SEQ ID NO:77] sequence, and WS
motif, [WQPWS; SEQ ID NO:72] sequence, was recognized in the
sequence. The comparison of the amino acid sequence of NR12 to that
of the known hematopoietin receptor, which were detected to have
homology in BlastX secondary search, are shown in FIG. 2. Based on
the above result, specific oligonucleotide primers were designed on
the exon sequence that were predicted in the AL109843 sequence, and
these primers were used in the 5'-RACE method and the 3'-RACE
method described later on.
(3) Design of Oligonucleotide Primers
[0185] As described above, exon sites were predicted on AL109843
sequences, and these were used to design the following
oligonucleotide primers specific for NR12. Three sense primers
(NR12-S1, NR12-S2, and NR12-S3; oriented downstream) and three
antisense primers (NR12-A1, NR12-A2, and NR12-A3; oriented
upstream) were synthesized using the ABI 394 DNA/RNA synthesizer
under a condition to attach a trityl group to the 5'-terminus.
Then, the products were purified using an OPC column (ABI #400771)
to obtain full-length primers.
TABLE-US-00003 NR12-S1; 5'- GCA ACA GTC AGA ATT CTA CTT GGA GCC -3'
(SEQ ID NO: 11) NR12-S2; 5'- CAT TAA GTA CGT ATT TCA AGT GAG ATG TC
-3' (SEQ ID NO: 12) NR12-S3; 5'- GGT ACT GGC AGC CTT GGA GTT CAC TG
-3' (SEQ ID NO: 13) NR12-A1; 5'- CAG TGA ACT CCA AGG CTG CCA GTA CC
-3' (SEQ ID NO: 14) NR12-A2; 5'- GAC ATC TCA CTT GAA ATA CGT ACT
TAA TG -3' (SEQ ID NO: 15) NR12-A3; 5'- GGC TCC AAG TAG AAT TCT GAC
TGT TGC -3' (SEQ ID NO: 16)
[0186] Above oligonucleotide primers, NR12-S1 and NR12-A3, NR12-S2
and NR12-A2, and NR12-S3 and NR12-A1 were designed to have
completely complementary sequence to each other.
(4) Cloning of N-Terminal cDNA by 5'-RACE Method
[0187] In order to isolate full-length cDNA of NR12, 5'-RACE PCR
was performed using NR12-A1 of (3) for primary PCR, and NR12-A2 of
(3) for secondary PCR, respectively. PCR experiment was performed
using Human Fetal Liver Marathon-Ready cDNA Library (Clontech
#7403-1) as the template, and Advantage cDNA Polymerase Mix
(Clontech #8417-1) on the thermal cycler (Perkin Elmer Gene Amp PCR
System 2400). Under the following conditions, as a result, PCR
products of two different sizes were obtained as shown in FIG.
3.
[0188] Condition of the primary PCR was as follows: 94.degree. C.
for 4 min, 5 cycles of "94.degree. C. for 20 sec, 72.degree. C. for
90 sec", 5 cycles of "94.degree. C. for 20 sec, 70.degree. C. for
90 sec", 28 cycles of "94.degree. C. for 20 sec, 68.degree. C. for
90 sec", 72.degree. C. for 3 min, and termination at 4.degree.
C.
[0189] Condition of the secondary PCR was as follows: 94.degree. C.
for 4 min, 5 cycles of "94.degree. C. for 20 sec, 70.degree. C. for
90 sec", 25 cycles of "94.degree. C. for 20 sec, 68.degree. C. for
90 sec", 72.degree. C. for 3 min, and termination at 4.degree.
C.
[0190] Two amplification products were obtained by the PCR and both
of them were subcloned into pGEM-T Easy vector (Promega #A1360),
and the nucleotide sequences were determined. The transformation of
the PCR product into the pGEM-T Easy vector was performed using T4
DNA ligase (Promega #1360) in a reaction at 4.degree. C. of 12
hours. Recombinants of the PCR products and pGEM-T Easy vector were
obtained by the transformation of E. coli DH5.alpha. strain (Toyobo
#DNA-903). Recombinants were selected by using Insert Check Ready
Blue (Toyobo #PIK-201). The nucleotide sequences were determined
using the BigDye Terminator Cycle Sequencing Ready Reaction Kit
(ABI/Perkin Elmer #4303154) and by analyzing with the ABI PRISM 377
DNA Sequencer. Nucleotide sequences of the whole insert fragment of
10 independent clones were determined. As a result, they were
divided into two groups, one consisting of 4 clones with a size of
1.3 kb, and the other consisting of 6 clones with a size of 1.0 kb,
based on the length of the base pairs and the differences in
sequence. However, the former 5'-RACE PCR products of 1.3 kb were
revealed to be non-specific PCR amplification products. This
sequence is derived from the minor band shown in FIG. 3. On the
other hand, the latter 5'-RACE PCR products of 1.0 kb were
recognized as partial nucleotide sequences of NR12 that resulted
from a correct PCR amplification reaction.
(5) Cloning of C-Terminal cDNA by 3'-RACE Method
[0191] To isolate the C-terminal sequence of a cDNA clone
corresponding to the full-length NR12.3'-RACE PCR was performed
using NR12-S1 primer of (3) for the primary PCR, and NR12-A2 of (3)
for secondary PCR, respectively. The PCR was performed under the
same condition as in the 5'-RACE above except the Human Thymus
Marathon-Ready cDNA Library (Clontech#7415-1) was used as the
template. More specifically, Advantage cDNA Polymerase Mix and the
Perkin Elmer Gene Amp PCR System 2400 thermalcycler was used in the
PCR experiment. Under the same PCR condition to those described in
(4), 3'-RACE amplification product showing an identical size of 750
bp was obtained as shown in FIG. 3. The obtained PCR product was
subcloned into the pGEM-T Easy vector as above to determine the
nucleotide sequence. The recombination of the PCR product into the
pGEM-T Easy vector was performed using T4 DNA ligase in a reaction
at 4.degree. C. for 12 hours. The recombinant of the PCR product
and pGEM-T Easy vector was obtained by transformation of E. coli
DH5.alpha. strain, and selection of the recombinant was done using
Insert Check Ready Blue as described above. The nucleotide sequence
was determined using the BigDye Terminator Cycle Sequencing Ready
Reaction Kit and the ABI PRISM 377 DNA Sequencer for analysis. The
nucleotide sequences of the whole insert fragment from 2
independent clones of genetic recombinants revealed that the clones
contain the C-terminal sequence of the full-length NR12 cDNA clone
having a poly A sequence.
[0192] Then, the nucleotide sequence determined by the 3'-RACE-PCR
and those determined by 5'-RACE-PCR in (4) were combined to finally
determine the whole nucleotide sequence of the cDNA clone encoding
the secretory form soluble receptor-like protein named NR12.1. The
determined nucleotide sequence of NR12.1 cDNA (SEQ ID NO:1) and the
amino acid sequence encoded by the sequence (SEQ ID NO:2) are shown
in FIG. 4.
(6) Cloning of a C-Terminal Splicing Variant by 3'-RACE Method
[0193] Although the NR12.1 clone isolated above had sufficient
feature of known hemopoietin receptors according to the result of
structural analysis, it did not possess a transmembrane region.
Therefore, it was inferred to encode a soluble receptor-like
protein as above-mentioned. Further, the present inventors
predicted the existence of splicing variants that have a
transmembrane region especially in the C-terminal region of the
transcription product of the present gene, and tried to isolate
NR12 cDNA clones by successive 3'-RACE method.
[0194] Thus, 3'-RACE PCR was performed using the above-mentioned
NR12-S2 primer of (3) for primary PCR, and NR12-S3 primer for
secondary PCR. Under the same PCR condition to those described in
(4) for 5'-RACE method except using Human Testis Marathon-Ready
cDNA Library (Clontech#7414-1) as the template. As a result,
multiple 3'-RACE PCR products with different sizes were obtained.
All of the obtained PCR products were subcloned into the pGEM-T
Easy vector as described above to determine the nucleotide
sequences. Nucleotide sequences of the whole insert fragments of 6
independent clones of genetic recombinants were determined. As a
result, one of these clones was found to be identical to NR12.1
determined above. The other 5 clones were possible to encode the
target transmembrane protein having transmembrane regions. That is,
the present inventors were able to confirm the existence of
splicing variants of NR12 as expected. Furthermore, the 5 cDNA
clones above showed differences in the C-terminal extracellular
region due to alternative splicing. Namely, two of these clones had
only a short intracellular region and were named NR12.2. On the
other hand, the other 3 clones had a long intracellular region.
These cDNA clones with a long ORF were named NR12.3, and were
distinguished from the above sequence, NR12.2.
[0195] Then, the nucleotide sequence determined by the 3'-RACE PCR
and those from the 5'-RACE PCR products in (4) were combined to
finally determine the whole nucleotide sequence of the cDNA clone
that encodes the transmembrane receptor protein. The nucleotide
sequence determined for NR12.2 cDNA (SEQ ID NO:3) and its amino
acid sequence (SEQ ID NO:4) are shown in FIG. 5. The nucleotide
sequence of NR12.3 cDNA (SEQ ID NO:5) and its amino acid sequence
(SEQ ID NO:6) are shown in FIGS. 6 and 7.
[0196] The exon site sequence was predicted in above (2) from the
splicing consensus sequence in RNA transcription (Hames, B. D. and
Glover, D. M., Transcription and Splicing (Oxford, IRL Press),
1988, p131-206) and not by using program such as genome analysis
software. According to the determination of the whole nucleotide
sequence of isolated cDNA clones, it was revealed that the exon
site predicted within the partial sequence of AL109843 shown in
FIG. 1 correspond completely to that observed in the actual
transcription of the NR12 gene. However, it was revealed that only
the transcription product of NR12.1 cDNA clone was one which
elongates to the 3'-untranslated region read through the identical
sequence as that of the genome structure without splicing after the
termination of WS exon.
(7) Structural Feature of NR12 and Prediction of its Function
[0197] As a result of the determination of the whole nucleotide
sequences of NR12.1, NR12.2 and NR12.3, it was revealed that they
are the transcription products having structural variety in the
C-terminus due to alternative splicing. The NR12.1 may encode a
secretory form soluble hemopoietin receptor-like protein consisting
of 337 amino acids according to its primary structure, while the
NR12.2 and NR12.3 may encode transmembrane hemopoietin receptor
proteins consisting of 428 and 629 amino acids, respectively. The
characteristics of each NR12 were as follows.
[0198] First, it is predicted that the sequence from the 1.sup.st
Met to the 23.sup.rd Gly in the common extracellular domain of
these clones is the typical secretion signal sequence. Herein, the
first Met is presumed to be the translation initiation site because
there exists an in-frame termination codon at the minus 32 position
from the 1.sup.st Met. Next, an Ig-like region exists in the region
from the 24.sup.th Gly to the 124.sup.th Pro residue. In addition,
it is predicted that the region from the 133.sup.rd Cys to the
144.sup.th Cys residue forms one of the loop structures which is a
ligand-binding site. Furthermore, the region from the 290.sup.th
Tyr to the 295.sup.th Arg residue corresponds to the highly
conserved YR motif, and a typical WS motif is also found at
residues from the 304.sup.th Trp to 308.sup.th Ser.
[0199] Herein, the NR12.1 encodes 29 amino acids after the WS motif
and the translation frame terminates at the next stop codon.
Therefore, the NR12.1 encodes a soluble hemopoietin receptor
protein that does not have a transmembrane domain. On the other
hand, the 26 amino acids following the conserved motif above from
the 352.sup.nd Gly to the 377.sup.th Asn residue in NR12.2 and
NR12.3 correspond to a typical transmembrane domain. The NR12.2 and
NR12.3 encode identical amino acid sequences to the 413.sup.th Gln
residue in the extracellular region. However, structural
differences exist in the C-terminal region following the 413.sup.th
Gln residue due to alternative splicing which connects them to
different exons. Namely, NR12.2 encodes 428 amino acids and the
translation frame is terminated at the next stop codon. Thus, it
has only a short intracellular region consisting of 51 amino acids.
On the other hand, NR12.3 encodes 629 amino acids and has an
intracellular region consisting of 252 amino acids. According to
the structural characteristics above, NR12 gene was recognized to
possess sufficient characteristics as novel hemopoietin receptor
proteins.
Example 2
Tissue Distribution Determination and Expression Pattern Analysis
of NR12 Gene by RT-PCR
[0200] mRNA was detected using the RT-PCR method to analyze the
expression distribution and the expression pattern of NR12.1 gene
in different human organs. NR12-PPD primer with the sequence below
was synthesized as a sense primer (downstream orientation) for the
RT-PCR analysis. NR12-A1 primer synthesized in Example 1 (3) was
used as the antisense primer (upstream orientation). The NR12-PPD
primer was synthesized and purified as in Example 1 (3). It was
expected that the common N-terminal region in all splice variants,
NR12.1, NR12.2 and NR12.3, are amplified and detected using these
primer sets (NR12-PPD and NR12-A1).
TABLE-US-00004 hNR12-PPD; 5'- CCG CCA GAT ATT CCT GAT GAA GTA ACC
-3' (SEQ ID NO: 17)
[0201] The templates used were Human Multiple Tissue cDNA (MTC)
Panel I (Clontech #K1420-1), Human MTC Panel II (Clontech
#K1421-1), Human Immune System MTC Panel (Clontech #K1426-1), and
Human Fetal MTC Panel (Clontech #K1425-1). PCR was performed using
Advantage cDNA Polymerase Mix (Clontech #8417-1) on a thermal
cycler (Perkin Elmer Gene Amp PCR System 2400). PCR was performed
by following condition to amplify the target gene: 94.degree. C.
for 4 min, 5 cycles of "94.degree. C. for 20 sec, 72.degree. C. for
1 min", 5 cycles of "94.degree. C. for 20 sec, 70.degree. C. for 1
min", 25 cycles of "94.degree. C. for 20 sec, 68.degree. C. for 1
min", 72.degree. C. for 3 min, and termination at 4.degree. C.
[0202] As shown in FIG. 8, strong expression of NR12 was observed
in the hematopoietic cell line tissue and immune system cell line
tissue such as adult spleen, thymus, lymph node, bone marrow, and
peripheral leukocyte. Expression was also detected in testis,
liver, lung, kidney, pancreas, and gastrointestinal tract such as
small intestine and colon. Moreover, NR12 gene expression was also
observed in all analyzed mRNA derived from human fatal tissues.
Performing PCR using human G3PDH primers under the above condition
and detecting the expression of the housekeeping gene G3PDH, it was
confirmed that the number of mRNA copies among the template mRNA
had been normalized.
[0203] The size of the RT-PCR amplification product was 561 bp,
which was consistent with the size calculated from the determined
nucleotide sequence of NR12 cDNA. Thus, the product was considered
to be the product of specific PCR amplification reaction. This was
further confirmed by Southern blotting as in the following, and the
possibility that the product was a non-specific PCR amplification
product was denied.
[0204] The analyses of expression distribution and expression
pattern of NR12 gene by RT-PCR revealed that the expression is
restricted to specific organs and tissues, and also that the amount
of expression varies greatly among organs. Taking all the result of
NR12 gene expression distribution together, the fact that
especially strong expression was detected in tissue considered
mainly to include immunocyte tissues and hematopoietic cells
suggest strongly the possibility that NR12 functions as a novel
hemopoietin receptor. Furthermore, the fact that the expression of
NR12 was also observed in other tissues suggests that NR12 can
regulate various physiological functions in vivo not only those in
the immune system and hematopoietic system.
[0205] Moreover, existence of splicing variants was recognized.
This strongly suggests that transcriptional regulation of the NR12
gene expression is strictly controlled by the transcriptional
regulation determining functional specificity, transcriptional
induction by exogenous stimulating factor, and regulation of
alternative splicing in specific cell types.
Example 3
Verification of the Specificity of RT-PCR Product by Southern
Blotting
[0206] In order to verify the specificity of amplification, the
RT-PCR amplified target gene product in Example 2 was subjected to
Southern blotting using NR12 specific cDNA fragment as a probe. At
the same time, the amount of RT-PCR product was quantitatively
detected by the strength of labeled signal to assess relative gene
expression levels among different human organs. The RT-PCR product
was electrophoresed on an agarose gel, blotted onto a charged nylon
membrane, Hybond N (+) (Amersham, cat #RPN303B), and was subjected
to hybridization. The 5'-RACE PCR product cDNA fragment
corresponding to the N-terminus of the NR12 obtained in Example 1
(4) was used as a probe specific to NR12. Probes were prepared
using the Mega Prime Kit (Amersham, cat #RPN1607), and labeled with
radioisotope, [.alpha.-.sup.32P] dCTP (Amersham, cat #AA0005).
Hybridization was performed using Express Hyb Hybridization
Solution (Clontech #8015-2), and after the prehybridization at
68.degree. C. for 30 min, heat-denatured labeled probe was added to
conduct hybridization at 68.degree. C. for 120 min. After
subsequent wash in (1) 1.times.SSC/0.1% SDS at room temperature for
5 min; (2) 1.times.SSC/0.1% SDS at 50.degree. C. for 30 min; and
(3) 0.1.times.SSC/0.1% SDS at 50.degree. C. for 30 min, the
membrane was exposed to Imaging Plate (FUJI #BAS-III), and NR12
specific signal was detected by the Image Analyzer (FUJIX, BAS-2000
II).
[0207] As shown in FIG. 9, all the amplified PCR products by the
RT-PCR above were verified as specific amplification products.
Furthermore, the result of quantification of relative expression
level among each tissue also supported above-mentioned assessment.
The detection method for target gene expression using RT-PCR and
Southern blotting in combination is known to have extremely high
sensitivity as compared to other methods for expression analysis.
Nevertheless, NR12 gene expression was not detected in adult heart,
skeletal muscle, adult brain, prostate, ovary, or placenta at
all.
Example 4
Northern Blot Analysis of NR12 Gene Expression
[0208] Northern blot analysis of NR12 gene expression was performed
to examine the expression pattern of NR12 gene in human organs and
human cancer cell lines, and to determine the size of NR12
transcripts. Human Multiple Tissue Northern (MTN) Blot (Clontech
#7760-1), Human MTN Blot II (Clontech #7759-1), Human MTN Blot III
(Clontech #7767-1), and Human Cancer Cell Line MTN Blot (Clontech
#7757-1) were used.
[0209] The cDNA fragment obtained by 5'-RACE in Example 1 (4) was
used as the probe. The probe was prepared using Mega Prime Kit and
radio-labeled with [.alpha.-.sup.32P]dCTP as in Example 3.
Hybridization was performed using Express Hybridization Solution,
and after prehybridization at 65.degree. C. for 30 min
heat-denatured labeled probe was added to conduct hybridization at
65.degree. C. for 16 hr. After subsequent wash in (1)
1.times.SSC/0.1% SDS at room temperature for 5 min; (2)
1.times.SSC/0.1% SDS at 48.degree. C. for 30 min; and (3)
0.5.times.SSC/0.1% SDS at 48.degree. C. for 30 min, the membrane
was exposed to an Imaging Plate as above, and an attempt to detect
NR12 specific signal was made using an Image Analyzer.
[0210] The method failed to detect any signal in any of the
examined human organs. This could be because Northern blotting has
a significant lower sensitivity than RT-PCR and thus failed to
detect mRNA with low expression level.
Example 5
Construction of an NR12 Ligand Screening System Using Growth
Factor-Dependent Cell Lines
[0211] Ligands that bind specifically to the protein of this
invention can be screened by the following step: (1) preparing a
chimeric receptor by ligating the extracellular domain of the
protein of this invention with the intracellular domain containing
the transmembrane domain of a hemopoietin receptor protein
comprising a known signal transduction ability; (2) expressing this
chimeric receptor on the cell surface of a suitable cell line,
preferably, a cell line that can survive and proliferate only under
the presence of a suitable factor (a growth factor-dependent cell
line); and (3) culturing the cell line by adding a material that is
expected to contain various growth factors, cytokines, or
hemopoietic factors. This method utilizes the fact that the
above-mentioned growth factor-dependent cell line only survives and
proliferates when a ligand specifically binding to the
extracellular domain of the protein of the invention exists within
the test material and is killed rapidly without the existence of
the growth factor. Known hemopoietic receptors are, for example,
the thrombopoietin receptor, erythropoietin receptor, G-CSF
receptor, gp130, etc. However, the partner of the chimeric receptor
used in the screening of the invention is not limited to these
known hemopoietic receptors, and any receptor may be used so long
as it contains the structure necessary for the signal transducing
activity in the cytoplasmic domain. IL-3-dependent cell lines, such
as Ba/F3 and FDC-P1, can be exemplified as growth factor-dependent
cell lines.
[0212] First, the cDNA sequence encoding the extracellular region
of NR12 (the amino acid sequence from the 1.sup.st Met to the
319.sup.th Gly) was amplified by PCR, and this DNA fragment was
bound in frame to the DNA fragments encoding the transmembrane
region and the intracellular region of a known hemopoietin receptor
to prepare a fusion sequence encoding a chimeric receptor. The TPO
receptor (Human MPL-P) was selected from the candidates described
above as the known partner hemopoietin receptor. The constructed
chimeric receptor sequence above was inserted into the plasmid
vector, pME18S/neo, which can be expressed in mammalian cells. A
schematic diagram of the structure of the constructed
pME18S/NR12-TPOR chimeric receptor is shown in FIG. 10. The
chimeric receptor-expressing vector was introduced into the growth
factor-dependent cell line Ba/F3, and was forced to express. Then,
stable gene-introduced cells were selected. The selection can be
done by utilizing the fact that the expression vector contains a
drug (neomycin) resistant gene, and thus, only gene-introduced
cells that obtained drug tolerance can be proliferated in the
culture containing the drug. Novel hematopoietin may be screened by
constructing a screening system that utilizes the ability of the
chimeric receptor-expressing cell lines to survive and proliferate
only under the existence of a ligand functionally binding
specifically to the NR12. In this case, the culture of the chimeric
receptor-expressing cell line is conducted in medium supplemented
with a material expected to include a target ligand in place of the
growth factor (IL-3, in this case) free medium described above.
Example 6
Construction of an Expression System of Secretary and Soluble
Recombinant NR12 Protein
[0213] Though rare, cell membrane-binding proteins except soluble
proteins can be envisaged as a ligand specifically binding to the
protein of the invention. In such cases, screening can be done by
labeling the protein containing only the extracellular domain of
the protein of the invention, or a fusion protein in which a
partial sequence of another soluble protein is added to the
extracellular domain of the present protein, and then, measuring
the binding with cells expected to express the ligand.
[0214] Examples of the former proteins containing only the
extracellular domain of the protein of the invention are, for
example, soluble receptor proteins artificially prepared by
inserting a stop codon to the N-terminal side of the transmembrane
domain, or NR12.1 that encodes the soluble type protein of NR12. On
the other hand, the latter proteins may be prepared by adding
labeling peptide sequences such as Fc site of immunoglobulins, and
FLAG peptide to the C-terminus of the extracellular domain of the
protein of the invention. These soluble labeled proteins can be
also used for the detection in the West-western blotting
method.
[0215] The present inventors selected a construction method as
follows: (1) cDNA sequence encoding the extracellular region of
NR12 (amino acid sequence from the 1.sup.st Met to the 319.sup.th
Gly) was amplified by PCR; and (2) FLAG peptide sequence was added
in frame to the C-terminus of the amplified DNA fragment to obtain
a sequence encoding the soluble targeted protein. The constructed
sequence was inserted into the plasmid vector, pCHO, which can be
expressed in mammalian cells. A schematic diagram of the structure
of the constructed pCHO/NR12-TPOR chimeric receptor is shown in
FIG. 10. This expression vector was introduced into mammalian
cells, CHO cells, and was forced to express. Then, stable
gene-introduced cells were selected. After confirming expression of
the soluble protein, the expression cells were cultured in large
scale. The recombinant protein secreted into the culture
supernatant can be immunoprecipitated using anti-FLAG peptide
antibody, and may be purified by affinity columns, etc.
[0216] The obtained recombinant protein can be applied not only for
the assay mentioned above, but also, for example, for detection of
specific biding activity within a material expected to contain a
target ligand by BIA-CORE system (Pharmacia). Thus, it is extremely
important for searching novel hemopoietins that can bind to
NR12.
Example 7
Reisolation of Human Full-Length NR12 CDS
(1) Design of Oligonucleotide Primers
[0217] The present inventors already had succeeded in isolating the
full-length cDNA of NR12 gene. However, the N-terminal sequence and
C-terminal sequence of the isolated target gene were isolated
separately due to the use of 5'-RACE and 3'-RACE method for the
cDNA isolation. Thus, the present inventors attempted to reisolate
NR12.2 and NR12.3 genes that contain continuous full-length coding
sequences.
[0218] First, a sense primer (NR12.2-MET) described below that
contains the start codon, Met sequence, with a common nucleotide
sequence to each cDNA clone of NR12 was designed. As the antisense
primers, NR12.2-STP and NR12.3-STP that contain a stop codon
specific to NR12.2 and NR12.3, respectively, were designed. The
primers were synthesis as in Example 1 (3). More specifically,
ABI's 394 DNA/RNA Synthesizer was used for the primer synthesis
under the condition where a trityl group is attached to the
5'-terminus. Then, the product was purified using and OPC column
(ABI#400771) to obtain full-length primers.
TABLE-US-00005 NR12.1-MET; 5'- ATG AAT CAG GTC ACT ATT CAA TGG -3'
(SEQ ID NO: 18) NR12.2-STP; 5'- GGA GTC CTC CTA CTT CAG CTT CCC -3'
(SEQ ID NO: 19) NR12.3-STP; 5'- TTG ATT TTG ACC ACA CAG CTC TAC -3'
(SEQ ID NO: 20)
(2) PCR Cloning
[0219] In order to isolate the full-length CDS of NR12, PCR cloning
was performed using NR12.1-MET primer of (1) as sense primer and
NR12.2-STP and NR12.3-STP primer as antisense primers,
respectively. Human Thymus Marathon-Ready cDNA Library
(Clontech#7415-1) was used as the template, and Advantage cDNA
Polymerase Mix (Clontech#8417-1) for the PCR experiment on a
thermal cycler (Perkin Elmer Gene Amp PCR System 2400) under the
condition described below. The PCR product of 1301 bp named NR12.4
was obtained using the primer set "NR12.1-MET and NR12.2-STP", and
that of 1910 bp named NR12.5 was obtained using the primer set
"NR12.1-MET and NR12.3-STP".
[0220] PCR was performed by a single cycle of "94.degree. C. for 4
min", 5 cycles of "94.degree. C. for 20 sec, 72.degree. C. for 90
sec", 5 cycles of "94.degree. C. for 20 sec, 70.degree. C. for 90
sec", 28 cycles of "94.degree. C. for 20 sec, 68.degree. C. for 90
sec", a single cycle of "72.degree. C. for 3 min", and was
terminated at 4.degree. C.
[0221] The obtained PCR products were subcloned into pGEM-T Easy
vectors (Promega #A1360) as in Example 1 (4), and the nucleotide
sequences were determined. Recombination of the PCR products into
the pGEM-T Easy vectors were performed using T4 DNA ligase (Promega
#1360) in a reaction at 4.degree. C. for 12 hours. The recombinant
of the PCR product and the pGEM-T Easy vector was obtained by
transformation of E. coli strain DH5.alpha. (Toyobo #DNA-903), and
Insert Check Ready Blue (Toyobo #PIK-201) was used for the
selection of the genetic recombinant. The nucleotide sequence was
determined using the BigDye Terminator Cycle Sequencing SF Ready
Reaction Kit (ABI/Perkin Elmer #4303150) and was analyzed by the
ABI PRISM 377 DNA Sequencer. The nucleotide sequence of the insert
fragments in respective recombinants of NR12.4 and NR12.5 were
analyzed, and the sequences of cDNA clones that may encode the
full-length CDS were determined.
[0222] As a result, it was revealed that NR12.4 contains the
full-length ORF of NR12.2 but not the 5'-untranslated region or
3'-untranslated region except the sequence derived from primers due
to the design of the used primers in the PCR. NR12.5 also contained
the full-length ORF of NR12.3 but not the 5'-untranslated region or
3'-untranslated region except the sequence derived from the
primers. The determined nucleotide sequence of NR12.4 and its amino
acid sequence are shown in FIGS. 11 and 12, and the determined
nucleotide sequence of NR12.5 and its amino acid sequence are shown
in FIGS. 13 and 14.
[0223] E. coli strain DH5.alpha. transfected with pGEM-T Easy
vector (pGEM/NR12.5CDS) that contains the NR12.5 cDNA of this
invention was deposited internationally on 31 Jul. 2000 as
follows.
[0224] Name and Address of the depositary institution
[0225] Depositary institution: National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology,
Ministry of International Trade and Industry.
[0226] Address: 1-1-3 Higashi, Tsukuba, Ibaraki 305-8566,
Japan.
[0227] Deposition date: 31 Jul. 2000
[0228] Accession No.: FERM BP-7259
Example 8
Cloning of Mouse NR12 Homologous Genomic Gene
(1) Preparation of a Probe Fragment of Human NR12
[0229] Aiming to analyze the genomic structure of mouse NR12 gene,
the present inventors performed plaque hybridization against the
mouse genomic DNA library. To perform heterologous cross
hybridization cloning against mouse genomic DNA library, probe
fragment of human NR12 cDNA was prepared. The insert fragment cut
out with Not I from the 5'-RACE product of human NR12 obtained in
Example 1 (4) was purified, and used as the probe fragment.
QIAquick Gel Extraction Kit (QIAGEN #28704) was used to extract and
purify the insert fragment from the agarose gel. The probe was
radiolabeled with [.alpha.-.sup.32P] dCTP using Mega Prime Kit as
in Example 3, and was used for plaque hybridization.
(2) Plaque Hybridization
[0230] Mouse 129SVJ strain Genomic DNA (Stratagene #946313)
constructed in Lambda FIX II was used as the library. A genomic
library of approximately 320 thousand plagues was developed in NZY
agar medium, and the plaques were blotted onto a Hybond N (+)
(Amersham #RPN303B) charged nylon membrane to conduct primary
screening. Perfect-Hyb Solution (Toyobo#HYB-101) was used for
hybridization, and after prehybridization at 60.degree. C. for 30
min, heat-denatured labeled probe was added, and hybridization was
conducted at 60.degree. C. for 16 hr. After subsequent wash in: (1)
1.times.SSC/0.1% SDS at room temperature for 5 min; (2)
1.times.SSC/0.1% SDS at 50.degree. C. for 30 min; and (3)
0.5.times.SSC/0.1% SDS at 50.degree. C. 30 min, the membrane was
exposed to an X-ray film (Hyperfilm MP: Amersham, #RPN8H) to detect
mouse NR21 positive plaques.
[0231] As a result, 6 independent positive or pseudo-positive
clones were obtained. The inventors succeeded in isolating plaques
of 2 independent NR12 positive clones by performing secondary
screening in a similar way to the primary screening against these 6
clones obtained by the primary screening. Lambda DNA of the
isolated plaque was prepared in large scale by plate-lysing method.
The insert fragments were cut out with restriction enzyme Sal I.
Analysis of their size revealed that the fragments were
approximately 18.5 kb and 16.0 kb, respectively.
INDUSTRIAL APPLICABILITY
[0232] The present invention provides novel hemopoietin receptor
proteins and DNA encoding same. The present invention also
provides: a vector into which the DNA has been inserted, a
transformant harboring the DNA, and a method for producing
recombinant proteins using the transformant. It further provides a
method of screening for a compound or a natural ligand that binds
to the protein. The protein of this invention is predicted to be
associated with the regulation of immune system and hematopoiesis.
Therefore, the proteins of this invention are expected to be useful
in understanding immune responses and fundamental features of
hematopoiesis in vivo. It is also expected that the proteins of the
present invention can be used in the diagnosis and treatment of
diseases related to immunity and hematopoiesis.
[0233] It is important to isolate unknown hematopoietic factors
that can bind to the NR12 molecule of this invention. The gene of
this invention is thought to be extremely useful in the screening
of such unknown factors. Furthermore, peptide libraries and
synthetic chemical materials may be searched to isolate and
identify agonists and antagonists that can functionally bind to the
NR12.
[0234] As described above, the NR12 gene is expected to provide a
useful source for obtaining unknown hematopoietic factors or
agonists that are capable of functionally binding to the receptor
protein encoded by the NR12 gene. It is expected that cellular
immunity and hematopoietic function in vivo will be enhanced by the
administration of such functionally binding substances or specific
antibodies that can activate the function of NR12 molecule to the
organism. Thus, the NR12 gene facilitates the development of drugs
for clinical application that promote proliferation or
differentiation of the immune cells or hematopoietic cells, or that
activates the function of immune cells. Such drugs may be used to
enhance cytotoxic immunity against specific types of tumor. It is
possible that NR12 is expressed in a restricted population of cells
in the hematopoietic tissues. Accordingly, anti-NR12 antibodies
would be useful in the isolation of such cell populations, which
may then be used in cell transplantation treatments.
[0235] On the other hand, NR12.1, a splice variant of NR12, may be
used as an inhibitor for the NR12 ligand, as a decoy type receptor.
Further, it is expected that by administering antagonists that can
bind functionally to the NR12 molecule, or other inhibitors, as
well as specific antibodies that can inhibit the molecular function
of NR12 to the organism, one can potentially suppress cellular
immunity or inhibit the proliferation of hematopoietic cells in
vivo. Thus, such inhibitors may be applied as drugs for clinical
application for use as, for example, proliferation inhibitors of
immune cell and hematopoietic cell, differentiation inhibitors,
immunosuppressive drugs, and anti-inflammatory drugs. Specifically,
such inhibitors may be used to suppress the onset of autoimmune
diseases arising from autoimmunity, or tissue rejection by the
immune system of the living body, the primary problem in
transplantation. Furthermore, the inhibitors may be effectively
used to treat diseases caused by such aberrant promotion of immune
response. Thus, the inhibitors may be used to treat a variety of
allergies that are specific to particular antigens, such as metal
and pollen.
Sequence CWU 1
1
7711784DNAHomo sapiensCDS(98)...(1108) 1atgacacagc caacaagggt
ggcagcctgg ctctgaagtg gaattatgtg cttcaaacag 60gttgaaagag ggaaacagtc
ttttcctgct tccagac atg aat cag gtc act att 115 Met Asn Gln Val Thr
Ile 1 5caa tgg gat gca gta ata gcc ctt tac ata ctc ttc agc tgg tgt
cat 163Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile Leu Phe Ser Trp Cys
His 10 15 20gga gga att aca aat ata aac tgc tct ggc cac atc tgg gta
gaa cca 211Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly His Ile Trp Val
Glu Pro 25 30 35gcc aca att ttt aag atg ggt gtg aat atc tct ata tat
tgc caa gca 259Ala Thr Ile Phe Lys Met Gly Val Asn Ile Ser Ile Tyr
Cys Gln Ala 40 45 50gca att aag aac tgc caa cca agg aaa ctt cat ttt
tat aaa aat ggc 307Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu His Phe
Tyr Lys Asn Gly 55 60 65 70atc aaa gaa aga ttt caa atc aca agg att
aat aaa aca aca gct cgg 355Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile
Asn Lys Thr Thr Ala Arg 75 80 85ctt tgg tat aaa aac ttt ctg gaa cca
cat gct tct atg tac tgc act 403Leu Trp Tyr Lys Asn Phe Leu Glu Pro
His Ala Ser Met Tyr Cys Thr 90 95 100gct gaa tgt ccc aaa cat ttt
caa gag aca ctg ata tgt gga aaa gac 451Ala Glu Cys Pro Lys His Phe
Gln Glu Thr Leu Ile Cys Gly Lys Asp 105 110 115att tct tct gga tat
ccg cca gat att cct gat gaa gta acc tgt gtc 499Ile Ser Ser Gly Tyr
Pro Pro Asp Ile Pro Asp Glu Val Thr Cys Val 120 125 130att tat gaa
tat tca ggc aac atg act tgc acc tgg aat gct ggg agg 547Ile Tyr Glu
Tyr Ser Gly Asn Met Thr Cys Thr Trp Asn Ala Gly Arg135 140 145
150ctc acc tac ata gac aca aaa tac gtg gta cat gtg aag agt tta gag
595Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val His Val Lys Ser Leu Glu
155 160 165aca gaa gaa gag caa cag tat ctc acc tca agc tat att aac
atc tcc 643Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser Ser Tyr Ile Asn
Ile Ser 170 175 180act gat tca tta caa ggt ggc aag aag tac ttg gtt
tgg gtc caa gca 691Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr Leu Val
Trp Val Gln Ala 185 190 195gca aac gca cta ggc atg gaa gag tca aaa
caa ctg caa att cac ctg 739Ala Asn Ala Leu Gly Met Glu Glu Ser Lys
Gln Leu Gln Ile His Leu 200 205 210gat gat ata gtg ata ctt tct gca
gcc gtc att tcc agg gct gag act 787Asp Asp Ile Val Ile Leu Ser Ala
Ala Val Ile Ser Arg Ala Glu Thr215 220 225 230ata aat gct aca gtg
ccc aag acc ata att tat tgg gat agt caa aca 835Ile Asn Ala Thr Val
Pro Lys Thr Ile Ile Tyr Trp Asp Ser Gln Thr 235 240 245aca att gaa
aag gtt tcc tgt gaa atg aga tac aag gct aca aca aac 883Thr Ile Glu
Lys Val Ser Cys Glu Met Arg Tyr Lys Ala Thr Thr Asn 250 255 260caa
act tgg aat gtt aaa gaa ttt gac acc aat ttt aca tat gtg caa 931Gln
Thr Trp Asn Val Lys Glu Phe Asp Thr Asn Phe Thr Tyr Val Gln 265 270
275cag tca gaa ttc tac ttg gag cca aac att aag tac gta ttt caa gtg
979Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile Lys Tyr Val Phe Gln Val
280 285 290aga tgt caa gaa aca ggc aaa agg tac tgg cag cct tgg agt
tca ctg 1027Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp Gln Pro Trp Ser
Ser Leu295 300 305 310ttt ttt cat aaa aca cct gaa aca ggt gag tgt
act tat ata ttt tat 1075Phe Phe His Lys Thr Pro Glu Thr Gly Glu Cys
Thr Tyr Ile Phe Tyr 315 320 325tct gtt ggg ctt ttc ttt ata tat ctt
ttc tgc tgagcacagt ggctcacgcc 1128Ser Val Gly Leu Phe Phe Ile Tyr
Leu Phe Cys 330 335tgtaattcca gcactttgag aggccaaggc aggaagattg
cttgagccta ggagtttgag 1188actggcctgg gcaacatggt gagaccctag
tctgtacaga aaaataataa ttattattag 1248cctgggtggt ggaatgcatt
tgtagtcgca gctacttggg aggctgaggt agtaggattg 1308cgtgagcccg
ggagtttgat gctgcagtga gctatgatca tcccactgct ctctagcctg
1368gaggaaagac caagaccctg tttcctaaaa agtttaaaac agccaggtgc
agtggcttat 1428gtctgtaatc ccagcacttt gggaggccaa ggtgggtgga
ttaccttagg tcaggacttc 1488aagacctcct cggccgacat ggtgaaaccc
tgtctctact aaaaatacga aaattagctg 1548ggcatggtgg caggtgcctg
taatctcagc tactcggaag gctgaggcag gaaaattgct 1608tgaacccaag
aagtggaggt tgcagtgaac tgagattgta ccaccgcact ccagcctggc
1668caagagagag agacttggtc tcaaaaaaaa ataaaaataa aaataataat
aataaataag 1728ttaaaaacaa aataaagcta caagataaaa aaaaaaaaaa
aaaaaaaaaa aaaaaa 17842337PRTHomo sapiens 2Met Asn Gln Val Thr Ile
Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15Leu Phe Ser Trp
Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His Ile Trp
Val Glu Pro Ala Thr Ile Phe Lys Met Gly Val Asn Ile 35 40 45Ser Ile
Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60His
Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile65 70 75
80Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His
85 90 95Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu
Thr 100 105 110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro
Asp Ile Pro 115 120 125Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser
Gly Asn Met Thr Cys 130 135 140Thr Trp Asn Ala Gly Arg Leu Thr Tyr
Ile Asp Thr Lys Tyr Val Val145 150 155 160His Val Lys Ser Leu Glu
Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser Tyr Ile Asn
Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190Leu Val
Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200
205Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Leu Ser Ala Ala Val
210 215 220Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr
Ile Ile225 230 235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val
Ser Cys Glu Met Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln Thr Trp
Asn Val Lys Glu Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val Gln Gln
Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val Phe Gln
Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln Pro Trp
Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Gly Glu305 310 315
320Cys Thr Tyr Ile Phe Tyr Ser Val Gly Leu Phe Phe Ile Tyr Leu Phe
325 330 335Cys31479DNAHomo sapiensCDS(98)...(1381) 3atgacacagc
caacaagggt ggcagcctgg ctctgaagtg gaattatgtg cttcaaacag 60gttgaaagag
ggaaacagtc ttttcctgct tccagac atg aat cag gtc act att 115 Met Asn
Gln Val Thr Ile 1 5caa tgg gat gca gta ata gcc ctt tac ata ctc ttc
agc tgg tgt cat 163Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile Leu Phe
Ser Trp Cys His 10 15 20gga gga att aca aat ata aac tgc tct ggc cac
atc tgg gta gaa cca 211Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly His
Ile Trp Val Glu Pro 25 30 35gcc aca att ttt aag atg ggt gtg aat atc
tct ata tat tgc caa gca 259Ala Thr Ile Phe Lys Met Gly Val Asn Ile
Ser Ile Tyr Cys Gln Ala 40 45 50gca att aag aac tgc caa cca agg aaa
ctt cat ttt tat aaa aat ggc 307Ala Ile Lys Asn Cys Gln Pro Arg Lys
Leu His Phe Tyr Lys Asn Gly 55 60 65 70atc aaa gaa aga ttt caa atc
aca agg att aat aaa aca aca gct cgg 355Ile Lys Glu Arg Phe Gln Ile
Thr Arg Ile Asn Lys Thr Thr Ala Arg 75 80 85ctt tgg tat aaa aac ttt
ctg gaa cca cat gct tct atg tac tgc act 403Leu Trp Tyr Lys Asn Phe
Leu Glu Pro His Ala Ser Met Tyr Cys Thr 90 95 100gct gaa tgt ccc
aaa cat ttt caa gag aca ctg ata tgt gga aaa gac 451Ala Glu Cys Pro
Lys His Phe Gln Glu Thr Leu Ile Cys Gly Lys Asp 105 110 115att tct
tct gga tat ccg cca gat att cct gat gaa gta acc tgt gtc 499Ile Ser
Ser Gly Tyr Pro Pro Asp Ile Pro Asp Glu Val Thr Cys Val 120 125
130att tat gaa tat tca ggc aac atg act tgc acc tgg aat gct ggg agg
547Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys Thr Trp Asn Ala Gly
Arg135 140 145 150ctc acc tac ata gac aca aaa tac gtg gta cat gtg
aag agt tta gag 595Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val His Val
Lys Ser Leu Glu 155 160 165aca gaa gaa gag caa cag tat ctc acc tca
agc tat att aac atc tcc 643Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser
Ser Tyr Ile Asn Ile Ser 170 175 180act gat tca tta caa ggt ggc aag
aag tac ttg gtt tgg gtc caa gca 691Thr Asp Ser Leu Gln Gly Gly Lys
Lys Tyr Leu Val Trp Val Gln Ala 185 190 195gca aac gca cta ggc atg
gaa gag tca aaa caa ctg caa att cac ctg 739Ala Asn Ala Leu Gly Met
Glu Glu Ser Lys Gln Leu Gln Ile His Leu 200 205 210gat gat ata gtg
ata ctt tct gca gcc gtc att tcc agg gct gag act 787Asp Asp Ile Val
Ile Leu Ser Ala Ala Val Ile Ser Arg Ala Glu Thr215 220 225 230ata
aat gct aca gtg ccc aag acc ata att tat tgg gat agt caa aca 835Ile
Asn Ala Thr Val Pro Lys Thr Ile Ile Tyr Trp Asp Ser Gln Thr 235 240
245aca att gaa aag gtt tcc tgt gaa atg aga tac aag gct aca aca aac
883Thr Ile Glu Lys Val Ser Cys Glu Met Arg Tyr Lys Ala Thr Thr Asn
250 255 260caa act tgg aat gtt aaa gaa ttt gac acc aat ttt aca tat
gtg caa 931Gln Thr Trp Asn Val Lys Glu Phe Asp Thr Asn Phe Thr Tyr
Val Gln 265 270 275cag tca gaa ttc tac ttg gag cca aac att aag tac
gta ttt caa gtg 979Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile Lys Tyr
Val Phe Gln Val 280 285 290aga tgt caa gaa aca ggc aaa agg tac tgg
cag cct tgg agt tca ctg 1027Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp
Gln Pro Trp Ser Ser Leu295 300 305 310ttt ttt cat aaa aca cct gaa
aca gtt ccc cag gtc aca tca aaa gca 1075Phe Phe His Lys Thr Pro Glu
Thr Val Pro Gln Val Thr Ser Lys Ala 315 320 325ttc caa cat gac aca
tgg aat tct ggg cta aca gtt gct tcc atc tct 1123Phe Gln His Asp Thr
Trp Asn Ser Gly Leu Thr Val Ala Ser Ile Ser 330 335 340aca ggg cac
ctt act tct gac aac aga gga gac att gga ctt tta ttg 1171Thr Gly His
Leu Thr Ser Asp Asn Arg Gly Asp Ile Gly Leu Leu Leu 345 350 355gga
atg atc gtc ttt gct gtt atg ttg tca att ctt tct ttg att ggg 1219Gly
Met Ile Val Phe Ala Val Met Leu Ser Ile Leu Ser Leu Ile Gly 360 365
370ata ttt aac aga tca ttc cga act ggg att aaa aga agg atc tta ttg
1267Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile Lys Arg Arg Ile Leu
Leu375 380 385 390tta ata cca aag tgg ctt tat gaa gat att cct aat
atg aaa aac agc 1315Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile Pro Asn
Met Lys Asn Ser 395 400 405aat gtt gtg aaa atg cta cag cca ggt gtg
gtg gtg tgc tcc tgt gat 1363Asn Val Val Lys Met Leu Gln Pro Gly Val
Val Val Cys Ser Cys Asp 410 415 420ccc agc tac ttg gga agc
tgaagtagga ggactgcttg agcccaggag 1411Pro Ser Tyr Leu Gly Ser
425tccaacacca gcttcacaac ataccaagac cctgtctcaa aaaaaaaaaa
aaaaaaaaaa 1471aaaaaaaa 14794428PRTHomo sapiens 4Met Asn Gln Val
Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15Leu Phe
Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His
Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Val Asn Ile 35 40
45Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu
50 55 60His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg
Ile65 70 75 80Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu
Glu Pro His 85 90 95Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His
Phe Gln Glu Thr 100 105 110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly
Tyr Pro Pro Asp Ile Pro 115 120 125Asp Glu Val Thr Cys Val Ile Tyr
Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140Thr Trp Asn Ala Gly Arg
Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val145 150 155 160His Val Lys
Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser
Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185
190Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys
195 200 205Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Leu Ser Ala
Ala Val 210 215 220Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro
Lys Thr Ile Ile225 230 235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu
Lys Val Ser Cys Glu Met Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln
Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val
Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val
Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln
Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val Pro305 310
315 320Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly
Leu 325 330 335Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp
Asn Arg Gly 340 345 350Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe
Ala Val Met Leu Ser 355 360 365Ile Leu Ser Leu Ile Gly Ile Phe Asn
Arg Ser Phe Arg Thr Gly Ile 370 375 380Lys Arg Arg Ile Leu Leu Leu
Ile Pro Lys Trp Leu Tyr Glu Asp Ile385 390 395 400Pro Asn Met Lys
Asn Ser Asn Val Val Lys Met Leu Gln Pro Gly Val 405 410 415Val Val
Cys Ser Cys Asp Pro Ser Tyr Leu Gly Ser 420 42552123DNAHomo
sapiensCDS(98)...(1984) 5atgacacagc caacaagggt ggcagcctgg
ctctgaagtg gaattatgtg cttcaaacag 60gttgaaagag ggaaacagtc ttttcctgct
tccagac atg aat cag gtc act att 115 Met Asn Gln Val Thr Ile 1 5caa
tgg gat gca gta ata gcc ctt tac ata ctc ttc agc tgg tgt cat 163Gln
Trp Asp Ala Val Ile Ala Leu Tyr Ile Leu Phe Ser Trp Cys His 10 15
20gga gga att aca aat ata aac tgc tct ggc cac atc tgg gta gaa cca
211Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly His Ile Trp Val Glu Pro
25 30 35gcc aca att ttt aag atg ggt gtg aat atc tct ata tat tgc caa
gca 259Ala Thr Ile Phe Lys Met Gly Val Asn Ile Ser Ile Tyr Cys Gln
Ala 40 45 50gca att aag aac tgc caa cca agg aaa ctt cat ttt tat aaa
aat ggc 307Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu His Phe Tyr Lys
Asn Gly 55 60 65 70atc aaa gaa aga ttt caa atc aca agg att aat aaa
aca aca gct cgg 355Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile Asn Lys
Thr Thr Ala Arg 75 80 85ctt tgg tat aaa aac ttt ctg gaa cca cat gct
tct atg tac tgc act 403Leu Trp Tyr Lys Asn Phe Leu Glu Pro His Ala
Ser Met Tyr Cys Thr 90 95 100gct gaa tgt ccc aaa cat ttt caa gag
aca ctg ata tgt gga aaa gac 451Ala Glu Cys Pro Lys His Phe Gln Glu
Thr Leu Ile Cys Gly Lys Asp 105 110 115att tct tct gga tat ccg cca
gat att cct gat gaa gta acc tgt gtc 499Ile Ser Ser Gly Tyr Pro Pro
Asp Ile Pro Asp Glu Val Thr Cys Val 120 125 130att tat gaa tat tca
ggc aac atg act tgc acc tgg aat gct ggg agg 547Ile Tyr Glu Tyr Ser
Gly Asn Met Thr Cys Thr Trp Asn Ala Gly Arg135 140 145 150ctc acc
tac ata gac aca aaa tac gtg gta
cat gtg aag agt tta gag 595Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val
His Val Lys Ser Leu Glu 155 160 165aca gaa gaa gag caa cag tat ctc
acc tca agc tat att aac atc tcc 643Thr Glu Glu Glu Gln Gln Tyr Leu
Thr Ser Ser Tyr Ile Asn Ile Ser 170 175 180act gat tca tta caa ggt
ggc aag aag tac ttg gtt tgg gtc caa gca 691Thr Asp Ser Leu Gln Gly
Gly Lys Lys Tyr Leu Val Trp Val Gln Ala 185 190 195gca aac gca cta
ggc atg gaa gag tca aaa caa ctg caa att cac ctg 739Ala Asn Ala Leu
Gly Met Glu Glu Ser Lys Gln Leu Gln Ile His Leu 200 205 210gat gat
ata gtg ata ctt tct gca gcc gtc att tcc agg gct gag act 787Asp Asp
Ile Val Ile Leu Ser Ala Ala Val Ile Ser Arg Ala Glu Thr215 220 225
230ata aat gct aca gtg ccc aag acc ata att tat tgg gat agt caa aca
835Ile Asn Ala Thr Val Pro Lys Thr Ile Ile Tyr Trp Asp Ser Gln Thr
235 240 245aca att gaa aag gtt tcc tgt gaa atg aga tac aag gct aca
aca aac 883Thr Ile Glu Lys Val Ser Cys Glu Met Arg Tyr Lys Ala Thr
Thr Asn 250 255 260caa act tgg aat gtt aaa gaa ttt gac acc aat ttt
aca tat gtg caa 931Gln Thr Trp Asn Val Lys Glu Phe Asp Thr Asn Phe
Thr Tyr Val Gln 265 270 275cag tca gaa ttc tac ttg gag cca aac att
aag tac gta ttt caa gtg 979Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile
Lys Tyr Val Phe Gln Val 280 285 290aga tgt caa gaa aca ggc aaa agg
tac tgg cag cct tgg agt tca ctg 1027Arg Cys Gln Glu Thr Gly Lys Arg
Tyr Trp Gln Pro Trp Ser Ser Leu295 300 305 310ttt ttt cat aaa aca
cct gaa aca gtt ccc cag gtc aca tca aaa gca 1075Phe Phe His Lys Thr
Pro Glu Thr Val Pro Gln Val Thr Ser Lys Ala 315 320 325ttc caa cat
gac aca tgg aat tct ggg cta aca gtt gct tcc atc tct 1123Phe Gln His
Asp Thr Trp Asn Ser Gly Leu Thr Val Ala Ser Ile Ser 330 335 340aca
ggg cac ctt act tct gac aac aga gga gac att gga ctt tta ttg 1171Thr
Gly His Leu Thr Ser Asp Asn Arg Gly Asp Ile Gly Leu Leu Leu 345 350
355gga atg atc gtc ttt gct gtt atg ttg tca att ctt tct ttg att ggg
1219Gly Met Ile Val Phe Ala Val Met Leu Ser Ile Leu Ser Leu Ile Gly
360 365 370aca ttt aac aga tca ttc cga act ggg att aaa aga agg atc
tta ttg 1267Thr Phe Asn Arg Ser Phe Arg Thr Gly Ile Lys Arg Arg Ile
Leu Leu375 380 385 390tta ata cca aag tgg ctt tat gaa gat att cct
aat atg aaa aac agc 1315Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile Pro
Asn Met Lys Asn Ser 395 400 405aat gtt gtg aaa atg cta cag gaa aat
agt gaa ctt atg aat aat aat 1363Asn Val Val Lys Met Leu Gln Glu Asn
Ser Glu Leu Met Asn Asn Asn 410 415 420tcc agt gag cag gtc cta tat
gtt gat ccc atg att aca gag ata aaa 1411Ser Ser Glu Gln Val Leu Tyr
Val Asp Pro Met Ile Thr Glu Ile Lys 425 430 435gaa atc ttc atc cca
gaa cac aag cct aca gac tac aag aag gag aat 1459Glu Ile Phe Ile Pro
Glu His Lys Pro Thr Asp Tyr Lys Lys Glu Asn 440 445 450aca gga ccc
ctg gag aca aga gac tac ccg caa aac tcg cta ttc gac 1507Thr Gly Pro
Leu Glu Thr Arg Asp Tyr Pro Gln Asn Ser Leu Phe Asp455 460 465
470aat act aca gtt gta tat att cct gat ctc aac act gga tat aaa ccc
1555Asn Thr Thr Val Val Tyr Ile Pro Asp Leu Asn Thr Gly Tyr Lys Pro
475 480 485caa att tca aat ttt ctg cct gag gga agc cat ctc agt aat
aat aat 1603Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser His Leu Ser Asn
Asn Asn 490 495 500gaa att act tcc tta aca ctt aaa cca cca gtt gat
tcc tta gac tca 1651Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro Val Asp
Ser Leu Asp Ser 505 510 515gga aat aat ccc agg tta caa aag cat cct
aat ttt gct ttt tct gtt 1699Gly Asn Asn Pro Arg Leu Gln Lys His Pro
Asn Phe Ala Phe Ser Val 520 525 530tca agt gtg aat tca cta agc aac
aca ata ttt ctt gga gaa tta agc 1747Ser Ser Val Asn Ser Leu Ser Asn
Thr Ile Phe Leu Gly Glu Leu Ser535 540 545 550ctc ata tta aat caa
gga gaa tgc agt tct cct gac ata caa aac tca 1795Leu Ile Leu Asn Gln
Gly Glu Cys Ser Ser Pro Asp Ile Gln Asn Ser 555 560 565gta gag gag
gaa acc acc atg ctt ttg gaa aat gat tca ccc agt gaa 1843Val Glu Glu
Glu Thr Thr Met Leu Leu Glu Asn Asp Ser Pro Ser Glu 570 575 580act
att cca gaa cag acc ctg ctt cct gat gaa ttt gtc tcc tgt ttg 1891Thr
Ile Pro Glu Gln Thr Leu Leu Pro Asp Glu Phe Val Ser Cys Leu 585 590
595ggg atc gtg aat gag gag ttg cca tct att aat act tat ttt cca caa
1939Gly Ile Val Asn Glu Glu Leu Pro Ser Ile Asn Thr Tyr Phe Pro Gln
600 605 610aat att ttg gaa agc cac ttc aat agg att tca ctc ttg gaa
aag 1984Asn Ile Leu Glu Ser His Phe Asn Arg Ile Ser Leu Leu Glu
Lys615 620 625tagagctgtg tggtcaaaat caatatgaga aagctgcctt
gcaatctgaa cttgggtttt 2044ccctgcaata gaaattgaat tctgcctctt
tttgaaaaaa atgtattcac atcccaaaaa 2104aaaaaaaaaa aaaaaaaaa
21236629PRTHomo sapiens 6Met Asn Gln Val Thr Ile Gln Trp Asp Ala
Val Ile Ala Leu Tyr Ile 1 5 10 15Leu Phe Ser Trp Cys His Gly Gly
Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His Ile Trp Val Glu Pro Ala
Thr Ile Phe Lys Met Gly Val Asn Ile 35 40 45Ser Ile Tyr Cys Gln Ala
Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60His Phe Tyr Lys Asn
Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile65 70 75 80Asn Lys Thr
Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 85 90 95Ala Ser
Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr 100 105
110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro
115 120 125Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met
Thr Cys 130 135 140Thr Trp Asn Ala Gly Arg Leu Thr Tyr Ile Asp Thr
Lys Tyr Val Val145 150 155 160His Val Lys Ser Leu Glu Thr Glu Glu
Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser Tyr Ile Asn Ile Ser Thr
Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190Leu Val Trp Val Gln
Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200 205Gln Leu Gln
Ile His Leu Asp Asp Ile Val Ile Leu Ser Ala Ala Val 210 215 220Ile
Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile225 230
235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser Cys Glu Met
Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn Val Lys Glu
Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr
Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val Phe Gln Val Arg Cys Gln
Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln Pro Trp Ser Ser Leu Phe
Phe His Lys Thr Pro Glu Thr Val Pro305 310 315 320Gln Val Thr Ser
Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu 325 330 335Thr Val
Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly 340 345
350Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser
355 360 365Ile Leu Ser Leu Ile Gly Thr Phe Asn Arg Ser Phe Arg Thr
Gly Ile 370 375 380Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu
Tyr Glu Asp Ile385 390 395 400Pro Asn Met Lys Asn Ser Asn Val Val
Lys Met Leu Gln Glu Asn Ser 405 410 415Glu Leu Met Asn Asn Asn Ser
Ser Glu Gln Val Leu Tyr Val Asp Pro 420 425 430Met Ile Thr Glu Ile
Lys Glu Ile Phe Ile Pro Glu His Lys Pro Thr 435 440 445Asp Tyr Lys
Lys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro 450 455 460Gln
Asn Ser Leu Phe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu465 470
475 480Asn Thr Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu Pro Glu Gly
Ser 485 490 495His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu
Lys Pro Pro 500 505 510Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg
Leu Gln Lys His Pro 515 520 525Asn Phe Ala Phe Ser Val Ser Ser Val
Asn Ser Leu Ser Asn Thr Ile 530 535 540Phe Leu Gly Glu Leu Ser Leu
Ile Leu Asn Gln Gly Glu Cys Ser Ser545 550 555 560Pro Asp Ile Gln
Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu 565 570 575Asn Asp
Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp 580 585
590Glu Phe Val Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro Ser Ile
595 600 605Asn Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn
Arg Ile 610 615 620Ser Leu Leu Glu Lys62571301DNAHomo
sapiensCDS(1)...(1284) 7atg aat cag gtc act att caa tgg gat gca gta
ata gcc ctt tac ata 48Met Asn Gln Val Thr Ile Gln Trp Asp Ala Val
Ile Ala Leu Tyr Ile 1 5 10 15ctc ttc agc tgg tgt cat gga gga att
aca aat ata aac tgc tct ggc 96Leu Phe Ser Trp Cys His Gly Gly Ile
Thr Asn Ile Asn Cys Ser Gly 20 25 30cac atc tgg gta gaa cca gcc aca
att ttt aag atg ggt atg aat atc 144His Ile Trp Val Glu Pro Ala Thr
Ile Phe Lys Met Gly Met Asn Ile 35 40 45tct ata tat tgc caa gca gca
att aag aac tgc caa cca agg aaa ctt 192Ser Ile Tyr Cys Gln Ala Ala
Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60cat ttt tat aaa aat ggc
atc aaa gaa aga ttt caa atc aca agg att 240His Phe Tyr Lys Asn Gly
Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile 65 70 75 80aat aaa aca aca
gct cgg ctt tgg tat aaa aac ttt ctg gaa cca cat 288Asn Lys Thr Thr
Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 85 90 95gct tct atg
tac tgc act gct gaa tgt ccc aaa cat ttt caa gag aca 336Ala Ser Met
Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr 100 105 110ctg
ata tgt gga aaa gac att tct tct gga tat ccg cca gat att cct 384Leu
Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro 115 120
125gat gaa gta acc tgt gtc att tat gaa tat tca ggc aac atg act tgc
432Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys
130 135 140acc tgg aat gct ggg aag ctc acc tac ata gac aca aaa tac
gtg gta 480Thr Trp Asn Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr
Val Val145 150 155 160cat gtg aag agt tta gag aca gaa gaa gag caa
cag tat ctc acc tca 528His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln
Gln Tyr Leu Thr Ser 165 170 175agc tat att aac atc tcc act gat tca
tta caa ggt ggc aag aag tac 576Ser Tyr Ile Asn Ile Ser Thr Asp Ser
Leu Gln Gly Gly Lys Lys Tyr 180 185 190ttg gtt tgg gtc caa gca gca
aac gca cta ggc atg gaa gag tca aaa 624Leu Val Trp Val Gln Ala Ala
Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200 205caa ctg caa att cac
ctg gat gat ata gtg ata cct tct gca gcc gtc 672Gln Leu Gln Ile His
Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val 210 215 220att tcc agg
gct gag act ata aat gct aca gtg ccc aag acc ata att 720Ile Ser Arg
Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile225 230 235
240tat tgg gat agt caa aca aca att gaa aag gtt tcc tgt gaa atg aga
768Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser Cys Glu Met Arg
245 250 255tac aag gct aca aca aac caa act tgg aat gtt aaa gaa ttt
gac acc 816Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn Val Lys Glu Phe
Asp Thr 260 265 270aat ttt aca tat gtg caa cag tca gaa ttc tac ttg
gag cca aac att 864Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu
Glu Pro Asn Ile 275 280 285aag tac gta ttt caa gtg aga tgt caa gaa
aca ggc aaa agg tac tgg 912Lys Tyr Val Phe Gln Val Arg Cys Gln Glu
Thr Gly Lys Arg Tyr Trp 290 295 300cag cct tgg agt tca ctg ttt ttt
cat aaa aca cct gaa aca gtt ccc 960Gln Pro Trp Ser Ser Leu Phe Phe
His Lys Thr Pro Glu Thr Val Pro305 310 315 320cag gtc aca tca aaa
gca ttc caa cat gac aca tgg aat tct ggg cta 1008Gln Val Thr Ser Lys
Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu 325 330 335aca gtt gct
tcc atc tct aca ggg cac ctt act tct gac aac aga gga 1056Thr Val Ala
Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly 340 345 350gac
att gga ctt tta ttg gga atg atc gtc ttt gct gtt atg ttg tca 1104Asp
Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser 355 360
365att ctt tct ttg att ggg ata ttt aac aga tca ttc cga act ggg att
1152Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile
370 375 380aaa aga agg atc tta ttg tta ata cca aag tgg ctt tat gaa
gat att 1200Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu
Asp Ile385 390 395 400cct aat atg aaa aac agc aat gtt gtg aaa atg
cta cag cca ggt gtg 1248Pro Asn Met Lys Asn Ser Asn Val Val Lys Met
Leu Gln Pro Gly Val 405 410 415gtg gtg tgc tcc tgt gat ccc agc tac
ttg gga agc tgaagtagga 1294Val Val Cys Ser Cys Asp Pro Ser Tyr Leu
Gly Ser 420 425ggactgc 13018428PRTHomo sapiens 8Met Asn Gln Val Thr
Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15Leu Phe Ser
Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His Ile
Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35 40 45Ser
Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55
60His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile65
70 75 80Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro
His 85 90 95Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln
Glu Thr 100 105 110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro
Pro Asp Ile Pro 115 120 125Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr
Ser Gly Asn Met Thr Cys 130 135 140Thr Trp Asn Ala Gly Lys Leu Thr
Tyr Ile Asp Thr Lys Tyr Val Val145 150 155 160His Val Lys Ser Leu
Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser Tyr Ile
Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190Leu
Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200
205Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val
210 215 220Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr
Ile Ile225 230 235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val
Ser Cys Glu Met Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln Thr Trp
Asn Val Lys Glu Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val Gln Gln
Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val Phe Gln
Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln Pro Trp
Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val Pro305 310 315
320Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu
325 330 335Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn
Arg Gly 340 345
350Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser
355 360 365Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr
Gly Ile 370 375 380Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu
Tyr Glu Asp Ile385 390 395 400Pro Asn Met Lys Asn Ser Asn Val Val
Lys Met Leu Gln Pro Gly Val 405 410 415Val Val Cys Ser Cys Asp Pro
Ser Tyr Leu Gly Ser 420 42591910DNAHomo sapiensCDS(1)...(1887) 9atg
aat cag gtc act att caa tgg gat gca gta ata gcc ctt tac ata 48Met
Asn Gln Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10
15ctc ttc agc tgg tgt cat gga gga att aca aat ata aac tgc tct ggc
96Leu Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly
20 25 30cac atc tgg gta gaa cca gcc aca att ttt aag atg ggt atg aat
atc 144His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn
Ile 35 40 45tct ata tat tgc caa gca gca att aag aac tgc caa cca agg
aaa ctt 192Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg
Lys Leu 50 55 60cat ttt tat aaa aat ggc atc aaa gaa aga ttt caa atc
aca agg att 240His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile
Thr Arg Ile 65 70 75 80aat aaa aca aca gct cgg ctt tgg tat aaa aac
ttt ctg gaa cca cat 288Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn
Phe Leu Glu Pro His 85 90 95gct tct atg tac tgc act gct gaa tgt ccc
aaa cat ttt caa gag aca 336Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro
Lys His Phe Gln Glu Thr 100 105 110ctg ata tgt gga aaa gac att tct
tct gga tat ccg cca gat att cct 384Leu Ile Cys Gly Lys Asp Ile Ser
Ser Gly Tyr Pro Pro Asp Ile Pro 115 120 125gat gaa gta acc tgt gtc
att tat gaa tat tca ggc aac atg act tgc 432Asp Glu Val Thr Cys Val
Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140acc tgg aat gct
ggg aag ctc acc tac ata gac aca aaa tac gtg gta 480Thr Trp Asn Ala
Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val145 150 155 160cat
gtg aag agt tta gag aca gaa gaa gag caa cag tat ctc acc tca 528His
Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170
175agc tat att aac atc tcc act gat tca tta caa ggt ggc aag aag tac
576Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr
180 185 190ttg gtt tgg gtc caa gca gca aac gca cta ggc atg gaa gag
tca aaa 624Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu
Ser Lys 195 200 205caa ctg caa att cac ctg gat gat ata gtg ata cct
tct gca gcc gtc 672Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro
Ser Ala Ala Val 210 215 220att tcc agg gct gag act ata aat gct aca
gtg ccc aag acc ata att 720Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr
Val Pro Lys Thr Ile Ile225 230 235 240tat tgg gat agt caa aca aca
att gaa aag gtt tcc tgt gaa atg aga 768Tyr Trp Asp Ser Gln Thr Thr
Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255tac aag gct aca aca
aac caa act tgg aat gtt aaa gaa ttt gac acc 816Tyr Lys Ala Thr Thr
Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270aat ttt aca
tat gtg caa cag tca gaa ttc tac ttg gag cca aac att 864Asn Phe Thr
Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285aag
tac gta ttt caa gtg aga tgt caa gaa aca ggc aaa agg tac tgg 912Lys
Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295
300cag cct tgg agt tca ctg ttt ttt cat aaa aca cct gaa aca gtt ccc
960Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val
Pro305 310 315 320cag gtc aca tca aaa gca ttc caa cat gac aca tgg
aat tct ggg cta 1008Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp
Asn Ser Gly Leu 325 330 335aca gtt gct tcc atc tct aca ggg cac ctt
act tct gac aac aga gga 1056Thr Val Ala Ser Ile Ser Thr Gly His Leu
Thr Ser Asp Asn Arg Gly 340 345 350gac att gga ctt tta ttg gga atg
atc gtc ttt gct gtt atg ttg tca 1104Asp Ile Gly Leu Leu Leu Gly Met
Ile Val Phe Ala Val Met Leu Ser 355 360 365att ctt tct ttg att ggg
ata ttt aac aga tca ttc cga act ggg att 1152Ile Leu Ser Leu Ile Gly
Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile 370 375 380aaa aga agg atc
tta ttg tta ata cca aag tgg ctt tat gaa gat att 1200Lys Arg Arg Ile
Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile385 390 395 400cct
aat atg aaa aac agc aat gtt gtg aaa atg cta cag gaa aat agt 1248Pro
Asn Met Lys Asn Ser Asn Val Val Lys Met Leu Gln Glu Asn Ser 405 410
415gaa ctt atg aat aat aat tcc agt gag cag gtc cta tat gtt gat ccc
1296Glu Leu Met Asn Asn Asn Ser Ser Glu Gln Val Leu Tyr Val Asp Pro
420 425 430atg att aca gag ata aaa gaa atc ttc atc cca gaa cac aag
cct aca 1344Met Ile Thr Glu Ile Lys Glu Ile Phe Ile Pro Glu His Lys
Pro Thr 435 440 445gac tac aag aag gag aat aca gga ccc ctg gag aca
aga gac tac ccg 1392Asp Tyr Lys Lys Glu Asn Thr Gly Pro Leu Glu Thr
Arg Asp Tyr Pro 450 455 460caa aac tcg cta ttc gac aat act aca gtt
gta tat att cct gat ctc 1440Gln Asn Ser Leu Phe Asp Asn Thr Thr Val
Val Tyr Ile Pro Asp Leu465 470 475 480aac act gga tat aaa ccc caa
att tca aat ttt ctg cct gag gga agc 1488Asn Thr Gly Tyr Lys Pro Gln
Ile Ser Asn Phe Leu Pro Glu Gly Ser 485 490 495cat ctc agt aat aat
aat gaa att act tcc tta aca ctt aaa cca cca 1536His Leu Ser Asn Asn
Asn Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro 500 505 510gtt gat tcc
tta gac tca gga aat aat ccc agg tta caa aag cat cct 1584Val Asp Ser
Leu Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys His Pro 515 520 525aat
ttt gct ttt tct gtt tca agt gtg aat tca cta agc aac aca ata 1632Asn
Phe Ala Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile 530 535
540ttt ctt gga gaa tta agc ctc ata tta aat caa gga gaa tgc agt tct
1680Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser
Ser545 550 555 560cct gac ata caa aac tca gta gag gag gaa acc acc
atg ctt ttg gaa 1728Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr
Met Leu Leu Glu 565 570 575aat gat tca ccc agt gaa act att cca gaa
cag acc ctg ctt cct gat 1776Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu
Gln Thr Leu Leu Pro Asp 580 585 590gaa ttt gtc tcc tgt ttg ggg atc
gtg aat gag gag ttg cca tct att 1824Glu Phe Val Ser Cys Leu Gly Ile
Val Asn Glu Glu Leu Pro Ser Ile 595 600 605aat act tat ttt cca caa
aat att ttg gaa agc cac ttc aat agg att 1872Asn Thr Tyr Phe Pro Gln
Asn Ile Leu Glu Ser His Phe Asn Arg Ile 610 615 620tca ctc ttg gaa
aag tagagctgtg tggtcaaaat caa 1910Ser Leu Leu Glu
Lys62510629PRTHomo sapiens 10Met Asn Gln Val Thr Ile Gln Trp Asp
Ala Val Ile Ala Leu Tyr Ile 1 5 10 15Leu Phe Ser Trp Cys His Gly
Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30His Ile Trp Val Glu Pro
Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35 40 45Ser Ile Tyr Cys Gln
Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60His Phe Tyr Lys
Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile65 70 75 80Asn Lys
Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 85 90 95Ala
Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu Thr 100 105
110Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro
115 120 125Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met
Thr Cys 130 135 140Thr Trp Asn Ala Gly Lys Leu Thr Tyr Ile Asp Thr
Lys Tyr Val Val145 150 155 160His Val Lys Ser Leu Glu Thr Glu Glu
Glu Gln Gln Tyr Leu Thr Ser 165 170 175Ser Tyr Ile Asn Ile Ser Thr
Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190Leu Val Trp Val Gln
Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200 205Gln Leu Gln
Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val 210 215 220Ile
Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile225 230
235 240Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser Cys Glu Met
Arg 245 250 255Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn Val Lys Glu
Phe Asp Thr 260 265 270Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr
Leu Glu Pro Asn Ile 275 280 285Lys Tyr Val Phe Gln Val Arg Cys Gln
Glu Thr Gly Lys Arg Tyr Trp 290 295 300Gln Pro Trp Ser Ser Leu Phe
Phe His Lys Thr Pro Glu Thr Val Pro305 310 315 320Gln Val Thr Ser
Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu 325 330 335Thr Val
Ala Ser Ile Ser Thr Gly His Leu Thr Ser Asp Asn Arg Gly 340 345
350Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met Leu Ser
355 360 365Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr
Gly Ile 370 375 380Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu
Tyr Glu Asp Ile385 390 395 400Pro Asn Met Lys Asn Ser Asn Val Val
Lys Met Leu Gln Glu Asn Ser 405 410 415Glu Leu Met Asn Asn Asn Ser
Ser Glu Gln Val Leu Tyr Val Asp Pro 420 425 430Met Ile Thr Glu Ile
Lys Glu Ile Phe Ile Pro Glu His Lys Pro Thr 435 440 445Asp Tyr Lys
Lys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro 450 455 460Gln
Asn Ser Leu Phe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu465 470
475 480Asn Thr Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu Pro Glu Gly
Ser 485 490 495His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu
Lys Pro Pro 500 505 510Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg
Leu Gln Lys His Pro 515 520 525Asn Phe Ala Phe Ser Val Ser Ser Val
Asn Ser Leu Ser Asn Thr Ile 530 535 540Phe Leu Gly Glu Leu Ser Leu
Ile Leu Asn Gln Gly Glu Cys Ser Ser545 550 555 560Pro Asp Ile Gln
Asn Ser Val Glu Glu Glu Thr Thr Met Leu Leu Glu 565 570 575Asn Asp
Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr Leu Leu Pro Asp 580 585
590Glu Phe Val Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro Ser Ile
595 600 605Asn Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn
Arg Ile 610 615 620Ser Leu Leu Glu Lys6251127DNAArtificial
SequenceArtificially synthesized primer sequence 11gcaacagtca
gaattctact tggagcc 271229DNAArtificial SequenceArtificially
synthesized primer sequence 12cattaagtac gtatttcaag tgagatgtc
291326DNAArtificial SequenceArtificially synthesized primer
sequence 13ggtactggca gccttggagt tcactg 261426DNAArtificial
SequenceArtificially synthesized primer sequence 14cagtgaactc
caaggctgcc agtacc 261529DNAArtificial SequenceArtificially
synthesized primer sequence 15gacatctcac ttgaaatacg tacttaatg
291627DNAArtificial SequenceArtificially synthesized primer
sequence 16ggctccaagt agaattctga ctgttgc 271727DNAArtificial
SequenceArtificially synthesized primer sequence 17ccgccagata
ttcctgatga agtaacc 271824DNAArtificial SequenceArtificially
synthesized primer sequence 18atgaatcagg tcactattca atgg
241924DNAArtificial SequenceArtificially synthesized primer
sequence 19gcagtcctcc tacttcagct tccc 242024DNAArtificial
SequenceArtificially synthesized primer sequence 20ttgattttga
ccacacagct ctac 24215PRTArtificial SequenceExemplary motif 21Trp
Ser Xaa Trp Ser 1 52215DNAArtificial SequenceExemplary motif
22tggagynnnt ggagy 1523360DNAHomo sapiensCDS(140)...(295)
23ttttatataa agaacacttt gttttcctag agtctagaag acagcttgga acataatagg
60tgttccatac atttctgcta aataaaatag ttgttttaaa agcacaccac attttattat
120tgttacccat ccattttag gtt aaa gaa ttt gac acc aat ttt aca tat gtg
172 Val Lys Glu Phe Asp Thr Asn Phe Thr Tyr Val 1 5 10caa cag tca
gaa ttc tac ttg gag cca aac att aag tac gta ttt caa 220Gln Gln Ser
Glu Phe Tyr Leu Glu Pro Asn Ile Lys Tyr Val Phe Gln 15 20 25gtg aga
tgt caa gaa aca ggc aaa agg tac tgg cag cct tgg agt tca 268Val Arg
Cys Gln Glu Thr Gly Lys Arg Tyr Trp Gln Pro Trp Ser Ser 30 35 40ctg
ttt ttt cat aaa aca cct gaa aca ggtgagtgta cttatatatt 315Leu Phe
Phe His Lys Thr Pro Glu Thr 45 50ttattctgtt gggcttttct ttatatatct
tttctgctga gcaca 3602452PRTHomo sapiens 24Val Lys Glu Phe Asp Thr
Asn Phe Thr Tyr Val Gln Gln Ser Glu Phe 1 5 10 15Tyr Leu Glu Pro
Asn Ile Lys Tyr Val Phe Gln Val Arg Cys Gln Glu 20 25 30Thr Gly Lys
Arg Tyr Trp Gln Pro Trp Ser Ser Leu Phe Phe His Lys 35 40 45Thr Pro
Glu Thr 502525PRTHomo sapiens 25Leu Glu Pro Asn Ile Lys Tyr Val Phe
Gln Val Arg Cys Gln Glu Thr 1 5 10 15Gly Lys Arg Tyr Trp Gln Pro
Trp Ser 20 252626PRTHomo sapiens 26Tyr Leu Glu Pro Asn Ile Lys Tyr
Val Phe Gln Val Arg Cys Gln Glu 1 5 10 15Thr Gly Lys Arg Tyr Trp
Gln Pro Trp Ser 20 252739PRTHomo sapiens 27Gln Gln Ser Glu Phe Tyr
Leu Glu Pro Asn Ile Lys Tyr Val Phe Gln 1 5 10 15Val Arg Cys Gln
Glu Thr Gly Lys Arg Tyr Trp Gln Pro Trp Ser Ser 20 25 30Leu Phe Phe
His Lys Thr Pro 352846PRTHomo sapiens 28Thr Asn Phe Thr Tyr Val Gln
Gln Ser Glu Phe Tyr Leu Glu Pro Asn 1 5 10 15Ile Lys Tyr Val Phe
Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr 20 25 30Trp Gln Pro Trp
Ser Ser Leu Phe Phe His Lys Thr Pro Glu 35 40 452934PRTHomo sapiens
29Leu Glu Pro Asn Ile Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr 1
5 10 15Gly Lys Arg Tyr Trp Gln Pro Trp Ser Ser Leu Phe Phe His Lys
Thr 20 25 30Pro Glu3026PRTHomo sapiens 30Leu Lys Pro Phe Thr Glu
Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 1 5 10 15Asp Gly Lys Gly
Tyr Trp Ser Asp Trp Ser 20 253126PRTHomo sapiens 31His Ile Asp Pro
Asn Val Asp Tyr Gln Phe Arg Val Cys Ala Arg Gly 1 5 10 15Asp Gly
Arg Gln Glu Trp Ser Pro Trp Ser 20 253241PRTHomo sapiens 32Gln Gln
Thr Glu Phe Lys Ile Leu Ser Leu His Pro Gly Gln Lys Tyr 1 5 10
15Leu Val Gln Val Arg Cys Lys Pro Asp His Gly Tyr Trp Ser Ala Trp
20 25 30Ser Pro Ala Thr Phe Ile Gln Ile Pro 35 403345PRTHomo
sapiens 33Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln Pro Ala Ala
Met Tyr 1 5 10 15Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr Phe
Lys Gly Phe Trp 20 25 30Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr
Pro Glu 35 40 453434PRTHomo sapiens 34Leu Asn Pro Tyr
Thr Leu Tyr Thr Phe Arg Ile Arg Cys Ser Thr Glu 1 5 10 15Thr Phe
Trp Lys Trp Ser Lys Trp Ser Asn Lys Lys Gln His Leu Thr 20 25 30Thr
Glu356PRTHomo sapiens 35Tyr Thr Phe Arg Ile Arg 1 5366PRTHomo
sapiens 36Tyr Val Phe Arg Ile Arg 1 5376PRTHomo sapiens 37Gln Glu
Phe Gln Leu Arg 1 5386PRTHomo sapiens 38Tyr Glu Phe Gln Ile Ser 1
5396PRTHomo sapiens 39Tyr Thr Leu Gln Ile Arg 1 5406PRTHomo sapiens
40Tyr Thr Phe Ala Val Arg 1 5416PRTHomo sapiens 41Tyr Arg Leu Gln
Leu Arg 1 5426PRTHomo sapiens 42Tyr Ala Val Gln Val Arg 1
5436PRTHomo sapiens 43Tyr Thr Val Gln Ile Arg 1 5446PRTHomo sapiens
44Tyr Arg Ala Arg Val Arg 1 5456PRTHomo sapiens 45Tyr Asp Val Gln
Val Arg 1 5466PRTHomo sapiens 46His Val Val Gln Leu Arg 1
5476PRTHomo sapiens 47Tyr Glu Ile Lys Val Arg 1 5486PRTHomo sapiens
48His Ala Val Arg Val Ser 1 5496PRTHomo sapiens 49Asn Thr Val Arg
Ile Arg 1 5506PRTHomo sapiens 50Tyr Glu Phe Gln Val Arg 1
5516PRTHomo sapiens 51Tyr Thr Phe Arg Val Arg 1 5526PRTHomo sapiens
52His Ser Val Lys Ile Arg 1 5536PRTHomo sapiens 53Tyr Ile Ile Gln
Val Ala 1 5546PRTHomo sapiens 54Tyr Leu Val Gln Val Arg 1
5556PRTHomo sapiens 55Tyr Phe Val Gln Val Arg 1 5566PRTHomo sapiens
56Tyr Gln Phe Arg Val Cys 1 5575PRTHomo sapiens 57Trp Ser Lys Trp
Ser 1 5585PRTHomo sapiens 58Trp Ser Asp Trp Ser 1 5595PRTHomo
sapiens 59Trp Ser Ala Trp Ser 1 5605PRTHomo sapiens 60Trp Ser Ser
Trp Ser 1 5615PRTHomo sapiens 61Trp Ser Asn Trp Ser 1 5625PRTHomo
sapiens 62Leu Ser Ala Trp Ser 1 5635PRTHomo sapiens 63Trp Ser Glu
Trp Ser 1 5645PRTHomo sapiens 64Trp Ser Thr Trp Ser 1 5655PRTHomo
sapiens 65Trp Ser Pro Trp Ser 1 5665PRTHomo sapiens 66Trp Ser Gly
Trp Ser 1 5675PRTHomo sapiens 67Trp Gly Glu Trp Ser 1 5685PRTHomo
sapiens 68Trp Ser Asp Trp Ala 1 5695PRTHomo sapiens 69Trp Ser Asn
Trp Lys 1 5705PRTHomo sapiens 70Trp Ser Met Trp Ser 1 5715PRTHomo
sapiens 71Trp Ser Gln Trp Ser 1 5725PRTHomo sapiens 72Trp Gln Pro
Trp Ser 1 5735PRTHomo sapiens 73Trp Ser Glu Trp Gly 1 5745PRTHomo
sapiens 74Thr Ser Gly Trp Ser 1 5755PRTHomo sapiens 75Trp Ser Arg
Trp Ser 1 5765PRTHomo sapiens 76Trp Ser Glu Gly Ser 1 5776PRTHomo
sapiens 77Tyr Val Phe Gln Val Arg 1 5
* * * * *