U.S. patent application number 11/940927 was filed with the patent office on 2009-08-06 for tsg-like gene.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Toshio Kitamura, Sumiyo Morita.
Application Number | 20090197317 11/940927 |
Document ID | / |
Family ID | 17233761 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090197317 |
Kind Code |
A1 |
Kitamura; Toshio ; et
al. |
August 6, 2009 |
TSG-Like Gene
Abstract
A gene encoding a novel protein that is homologous to Drosophila
TSG was isolated from a cDNA library derived from the AGM region of
mouse embryos by using an originally developed cloning method
specific to a gene encoding a membrane secretory protein. This gene
is useful in developing drugs that regulate hematopoietic stem cell
generation, immune and hematopoietic functions, etc.
Inventors: |
Kitamura; Toshio; (Tokyo,
JP) ; Morita; Sumiyo; (Tokyo, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Chugai Seiyaku Kabushiki
Kaisha
|
Family ID: |
17233761 |
Appl. No.: |
11/940927 |
Filed: |
November 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11079947 |
Mar 15, 2005 |
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11940927 |
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10092925 |
Mar 6, 2002 |
6890735 |
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11079947 |
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PCT/JP00/06050 |
Sep 6, 2000 |
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10092925 |
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Current U.S.
Class: |
435/188 ;
530/350; 530/387.3 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/475 20130101 |
Class at
Publication: |
435/188 ;
530/350; 530/387.3 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C07K 14/00 20060101 C07K014/00; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 1999 |
JP |
11/252190 |
Claims
1. A substantially purified polypeptide comprising residues 25-222
of SEQ ID NO:2.
2. The polypeptide of claim 1, wherein the polypeptide further
comprises an initiator methionine or a signal peptide.
3. The polypeptide of claim 1, wherein the polypeptide consists of
residues 25-222 of SEQ ID NO:2 plus an initiator methionine or a
signal peptide.
4. The polypeptide of claim 1, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:2.
5. The polypeptide of claim 1, wherein the polypeptide consists of
the amino acid sequence of SEQ ID NO:2.
6. The polypeptide of claim 1, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:2 fused to a second amino acid
sequence.
7. The polypeptide of claim 1, wherein the polypeptide comprises
the amino acid sequence of residues 25-222 of SEQ ID NO:2 fused to
a second amino acid sequence.
8. The polypeptide of claim 7, wherein the second amino acid
sequence is a peptide selected from the group consisting of
glutathione S-transferase, FLAG, six histidine residues, 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, immunoglobulin
constant region, .beta.-galactosidase, Green Fluorescent Protein
(GFP), and maltose binding protein.
9. The polypeptide of claim 7, wherein the polypeptide comprises an
initiator methionine.
10. The polypeptide of claim 7, wherein the polypeptide further
comprises residues 1-24 of SEQ ID NO:2.
11. The polypeptide of claim 7, wherein the polypeptide comprises a
signal sequence.
12. A substantially purified polypeptide that (a) has at least 95%
identity to the amino acid sequence of SEQ ID NO:2, and (b) binds
to BMP2/4.
13. The polypeptide of claim 12, wherein the polypeptide has at
least 98% identity to the amino acid sequence of SEQ ID NO:2.
14. The polypeptide of claim 12, wherein the polypeptide has at
least 99% identity to the amino acid sequence of SEQ ID NO:2.
15. A substantially purified polypeptide comprising the amino acid
sequence of SEQ ID NO:2 or a fragment thereof, wherein the fragment
is at least 40% of the length of the sequence shown as SEQ ID NO:2,
and the fragment binds to BMP2/4.
16. A substantially purified polypeptide comprising the amino acid
sequence of SEQ ID NO:2 in which 5 or fewer amino acids are
substituted, deleted, and/or inserted, and the polypeptide binds to
BMP2/4.
Description
[0001] This application is a continuation of U.S. Ser. No.
11/079,947, filed Mar. 15, 2005, which is a divisional application
of U.S. Ser. No. 10/092,925, filed Mar. 6, 2002, now U.S. Pat. No.
6,890,735, which is a continuation-in-part of International Patent
Application No. PCT/JP00/06050, filed Sep. 6, 2000, which claims
priority to Japanese Patent Application No. 11-252190, filed Sep.
6, 1999. The disclosures of all of these applications are herein
incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a novel TSG-like protein
and its gene derived from the AGM region of mouse embryos.
BACKGROUND
[0003] In the early fetal period of mice, hematopoiesis is carried
out in the yolk sac and fetal liver. Hematopoiesis in the yolk sac
is referred to as fetal hematopoiesis during which primarily
nucleated fetal erythrocytes are produced. On the other hand,
hematopoiesis in the fetal liver is referred to as adult
hematopoiesis during which all lines of blood cells are produced
with the exception of nucleated fetal erythrocytes.
[0004] Although the activity that causes the production of all
lines of blood cells, that is the long-term repopulating
hematopoietic stem cell (LTR-HSC) activity of hematopoietic stem
cells, is not detected in fetal hematopoiesis, it is detected in
adult hematopoiesis. It is now thought that the cells having this
LTR-HSC activity are actually produced not initially in the liver,
but in the aorta-gonad-mesonephros (AGM) region at day 10-11 of
embryogenesis. During this period, these cells are thought to also
proliferate in this AGM region, after which they migrate to the
fetal liver (Medvinsky et al., Cell 86:897-906). Thus, a gene that
is important for the generation of hematopoietic stem cells may be
expressed in this AGM region.
SUMMARY
[0005] The present invention provides a novel TSG-like protein and
its gene derived from the AGM region of mouse embryos. In addition,
the present invention also provides a vector into which the gene is
inserted, a host cell carrying the vector, and an antibody that
binds to the protein. Moreover, the present invention provides a
method for screening compounds, such as receptors, that bind to the
protein by using the protein.
[0006] The present inventors screened cDNA that encode
secretory/membrane proteins to search for a gene having a novel
signal sequence from the AGM region of mouse embryos, using poly(A)
RNA derived from this AGM region as the starting material, and an
originally-developed signal sequence trap (SST) method (Japanese
Patent Application No. Hei 9-324912). As a result, the present
inventors succeeded in isolating a gene that encodes a novel
protein homologous to Drosophila TSG gene. TSG gene is one of the
dorsal determining factors of an embryo, and is known to determine
differentiation of dorsal midline cells due to interaction with DPP
(the counterpart of BMP2/4) (Mason et al., Genes and Development
8:1489-1501). Since TSG protein has been reported to bind to BMP
(Oelgeschlager et al., Nature 405:757-763, 2000), the isolated
TSG-like gene, which is structurally similar to the TSG gene, is
predicted to interact with BMP2/4. In addition, the fact that this
TSG-like gene was isolated from the AGM region of mouse embryos
suggests its involvement in the generation of hematopoietic stem
cells. Thus, the TSG-like protein of the present invention is
useful as a tool for purifying and screening factors involved in
the generation of hematopoietic stem cells, and the screening of
drug candidate compounds for immune and hematopoietic
system-related diseases.
[0007] The present invention relates to a novel TSG-like protein,
its gene, as well as the production and uses of the protein and
gene. More specifically, the present invention relates to:
[0008] (1) a DNA according to any one of (a) to (d):
[0009] (a) a DNA encoding a protein comprising the amino acid
sequence of SEQ ID NO:2,
[0010] (b) a DNA comprising the coding region of the nucleotide
sequence of SEQ ID NO:1,
[0011] (c) a DNA comprising an amino acid sequence in which one or
more amino acids of the amino acid sequence of SEQ ID NO:2 has been
substituted, deleted, inserted and/or added, wherein said DNA
encodes a protein that is functionally equivalent to the protein
comprising the amino acid sequence of SEQ ID NO:2, and,
[0012] (d) a DNA hybridizing to a DNA that comprises the nucleotide
sequence of SEQ ID NO:1 under stringent conditions, and, encodes a
protein functionally equivalent to the protein comprising the amino
acid sequence of SEQ ID NO:2;
[0013] (2) a DNA encoding a partial peptide of the protein
comprising the amino acid sequence of SEQ ID NO:2;
[0014] (3) a vector into which the DNA according to (1) or (2) has
been inserted;
[0015] (4) a transformant carrying the DNA according to (1) or (2)
or the vector according to (3);
[0016] (5) a protein or peptide encoded by the DNA according to (1)
or (2);
[0017] (6) a method for producing the protein or peptide according
to (5), comprising the steps of culturing the transformed cell
according to (4), and recovering the expressed protein from said
cell or the culture supernatant;
[0018] (7) an antibody against the protein according to (5);
[0019] (8) a oligonucleotide that
[0020] hybridizes to the DNA comprising of the nucleotide sequence
of SEQ ID NO:1, or the complementary strand thereof, and,
[0021] comprises at least 15 nucleotides;
[0022] (9) a method of screening for a compound having the activity
of binding to the protein according to (5), comprising the steps
of:
[0023] (a) contacting a test sample with the protein or partial
peptide according to (5), and,
[0024] (b) selecting a compound having an activity of binding to
the protein or partial peptide according to (5); and,
[0025] (10) a compound isolated using a method as set forth in (9),
having an activity of binding to the protein according to (5).
[0026] The present invention relates to a novel protein that is
homologous to the Drosophila TSG gene. A mouse-derived cDNA
nucleotide sequence isolated by the present inventors is shown in
SEQ ID NO:1, while the amino acid sequence of the protein encoded
by the cDNA is shown in SEQ ID NO:2. This protein has a signal
sequence at its N terminus, and is homologous to TSG protein, a
dorsal determining factor of the Drosophila embryo. As a result of
Northern blot analysis of mRNA derived from mouse tissues, a signal
of about 4.0 kb was observed in the heart, lung, liver, and kidney.
In addition, this signal was also confirmed to be expressed in 9,
10, 11, 12, and 13-day viviparity. The isolation from the AGM
region of the embryo, expression in early embryos, homology to TSG
protein and presumed interaction with BMP2/4, the fact that BMP2/4
is required for the differentiation of blood cell lines, the fact
that TSG protein binds to BMP to promote signaling activity of BMP
(Oelgeschlager et al., Nature 405:757-763, 2000), all suggest that
this protein may be involved in the differentiation of
hematopoietic cells as well as bone formation, and so forth. Thus,
this protein can be utilized as a tool for purifying and cloning
proteins related to hematopoietic stem cell formation, bone
formation, and so forth, and for screening drug candidate compounds
as therapeutic agents for immune and hematopoietic system-related
diseases, bone formation-related diseases, and such.
[0027] 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.
[0028] Accordingly, the invention includes a polypeptide having a
sequence shown as SEQ ID NO:2. The invention also includes a
polypeptide, or fragment thereof, that differs from the
corresponding sequence shown as SEQ ID NO:2. 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, 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 and has at least one TSG-like
function or activity described herein. 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 and have at least one TSG-like
function or activity described herein. Or alternatively, the
fragment can be merely an immunogenic fragment.
[0029] In addition, the present invention also includes proteins
that are functionally equivalent to the protein described in SEQ ID
NO:2. Such proteins include, for example, homologous proteins of
other organisms corresponding to the protein described in SEQ ID
NO:2, as well as mutants of the protein. In the present invention,
the term "functionally equivalent" means that the target protein
has an activity for rescuing aberrations in the differentiation of
dorsal midline cells when injected into a TSG mutant of Drosophila,
or an activity that regulates embryo development (for example,
dorsoventral induction capability) when injected into Xenopus eggs.
In addition, the protein of the present invention is also suggested
to have the function of promoting the signaling activity of BMP
(bone morphogenetic protein; DPP) by binding with BMP
(Oelgeschlager et al., Nature 405:757-763, 2000).
[0030] One method for isolating such proteins well known to those
skilled in the art is to introduce mutations into the proteins. For
example, one skilled in the art can prepare proteins functionally
equivalent to the protein of SEQ ID NO:2 by introducing appropriate
mutations into the amino acid of SEQ ID NO:2, by using
site-specific 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). Mutation of amino acids may occur in nature,
too. The protein of the present invention also includes a protein
comprising the amino acid sequence of SEQ ID NO:2 in which one or
more amino acids are mutated, wherein the resulting "mutant"
protein is functionally equivalent to the protein of SEQ ID NO:2.
In such a mutant protein, the number of the amino acids mutated are
considered to be usually 30 residues or less, preferably 15
residues or less, more preferably 5 residues or less, and still
preferably, 3 residues or less.
[0031] The mutated amino acid residue is preferably mutated into an
amino acid that allows the properties of the amino acid side-chain
to be conserved. Examples of properties of amino acid side chains
include: 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 (Q 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
nucleotide-containing side-chain (R, K, H); and an
aromatic-containing side-chain (H, F, Y, W) (the letters within the
parentheses indicate the one-letter codes of amino acids).
[0032] It is well known that a protein having a deletion, addition,
and/or substitution of one or more amino acid residues in the
sequence of the protein can retain the original 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).
[0033] A protein having the amino acid sequence of SEQ ID NO:2, to
which one or more amino acid residues have been added, is
exemplified by a fusion protein containing the protein of SEQ ID
NO:2. Fusion proteins, in which the protein listed in SEQ ID NO:2,
or its partial peptide is fused to other peptides or proteins, are
included in the present invention. Fusion proteins can be made
using well-known techniques by linking the DNA encoding the protein
of the invention in frame with the DNA encoding another peptide or
protein, followed by inserting the DNA into an expression vector,
and expressing it in a host. There is no restriction as to the
peptides or proteins to be fused to the protein of the present
invention.
[0034] Other known peptides that can be used for fusion with the
protein of the present invention include, for example, FLAG (Hopp
et al., BioTechnology 6:1204-1210, 1988), 6.times.His comprised 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, etc. In
addition, examples of other proteins used for fusion with the
protein of the present invention include, for example, GST
(glutathione-S-transferase), HA (influenza agglutinin),
immunoglobulin constant region, .beta.-galactosidase, MBP (maltose
binding protein), etc.
[0035] Fusion proteins can be prepared by fusing DNA encoding these
commercially available peptides or proteins with DNA encoding the
protein of the present invention and expressing the prepared fused
DNA.
[0036] An alternative method for isolating functionally equivalent
proteins known to those skilled in the art is, for example, a
method utilizing the hybridization technique (Sambrook et al.,
Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press,
1989). Generally, one skilled in the art can isolate DNAs highly
homologous to the whole or part of the DNA sequence encoding the
protein of SEQ ID NO:2 or 4 (SEQ ID NO:1 or 3, respectively), and
then isolate a DNA that codes for a protein functionally equivalent
to the protein of SEQ ID NO:2 or 4 from the DNA isolated. The
proteins of the present invention thus include proteins encoded by
DNA that hybridize with the whole or part of the DNA sequence
encoding the protein of SEQ ID NO:2 or 4, wherein the proteins are
functionally equivalent to the protein of SEQ ID NO:2 or 4. These
proteins include homologues from mammals except mice (for example,
a protein encoded by a human gene).
[0037] Hybridization for isolating a DNA encoding a functionally
equivalent protein can be carried out under the stringent
conditions of, for example, 10% formamide, 5.times.SSPE,
1.times.Denhardt's solution, and 1.times. salmon sperm DNA. More
preferable (more stringent) conditions are, 25% formamide,
5.times.SSPE, 1.times.Denhardt's solution, and 1.times. salmon
sperm DNA, and even more preferable (even more stringent)
conditions are, 50% formamide, 5.times.SSPE, 1.times.Denhardt's
solution, and 1.times. salmon sperm DNA. However, several factors
are thought to influence the stringency of hybridization other than
the above-described formamide concentration, and one skilled in the
art can suitably select these factors to accomplish a similar
stringency. Also, instead of hybridization, it is also possible to
isolate a DNA encoding a functionally equivalent protein by a gene
amplification method such as PCR using a portion of the DNA
encoding the protein (SEQ ID NO:1) as a primer.
[0038] The proteins encoded by DNA isolated by hybridization or
gene amplifying techniques having functions equivalent to the
protein of SEQ ID NO:2 are usually highly homologous to the protein
of SEQ ID NO:2 at the amino acid sequence level. The protein of the
present invention also includes a protein that is functionally
equivalent to the protein of SEQ ID NO:2 and has a high homology to
the amino acid sequence indicated in SEQ ID NO:2. "High homology"
refers to an amino acid sequence identity of 40% or more,
preferably 50% or more, and more preferably 60% or more. The
algorithm described in the literature (Wilbur et al., Proc. Natl.
Acad. Sci. USA 80:726-730, 1983) may be used for determining
protein homology.
[0039] The protein of the present invention may be different in the
amino acid sequence, molecular weight, isoelectric point, presence
or absence of a sugar chain, or the form of the sugar chain, and so
forth depending on the cells that produce it, the host, or
purification process (described later). However, such proteins are
included in the present invention provided the resulting protein is
functionally equivalent to the protein described in SEQ ID NO:2.
For example, when the protein of the present invention is expressed
in prokaryotic cells, for example, E. Coli, a methionine residue is
added to the N-terminus of the amino acid sequence of the original
protein. Alternatively, when expressed in eukaryotic cells, for
example, mammalian cells, the N-terminus signal sequence is
removed. The protein of the present invention also includes such
proteins. As a result of analyzing the amino acid sequence of the
protein of the present invention, the signal sequence was estimated
to extend from Met at position 1 to Ser at position 24 in the amino
acid sequence of SEQ ID NO:2. Thus, the present invention includes
proteins comprising amino acids from Cys at position 25 to Phe at
position 222 in the amino acid sequence described in SEQ ID
NO:2.
[0040] The protein of the present invention can be prepared as a
recombinant protein or as a naturally-occurring protein by methods
known to those skilled in the art. If it is a recombinant protein,
the protein is secreted extracellularly as, for example, a soluble
protein. Subsequently, the culture supernatant of the cells can be
recovered, concentrated and then purified by chromatography
utilizing ion exchange, reverse phase, or gel filtration
chromatography, or by affinity chromatography using a column in
which an antibody against the protein of the present invention is
immobilized, or by a combination of these columns. Alternatively,
the protein of the invention can be prepared by expressing the
protein in host cells (e.g., animal cells or E. coli) as a fusion
protein with glutathione S transferase protein, or a recombinant
protein with multiple histidine residues. The expressed protein can
be purified using a glutathione column or nickel column.
Subsequently, if necessary, regions of the fusion protein (apart
from the desired protein) can be digested and removed with thrombin
or factor Xa, etc. The natural form of the protein of the invention
can be isolated by, for example, purifying a cell extract
containing the protein with an affinity column to which the
antibody of the present invention described below is bound.
[0041] The present invention also includes partial peptides of the
protein of the present invention. A partial peptide of the present
invention comprises an amino acid sequence of at least seven amino
acids, preferably eight or more amino acids, and more preferably
nine or more amino acids. The partial peptide can be used for, for
example, production of an antibody against the protein of the
present invention, screening of compounds that bind to the present
protein, screening of receptors of the present protein, or
preparation of a competition inhibitor of the present protein. In
addition, present invention includes partial peptides having, for
example, the ability to bind to a receptor, but not the ability to
activate the receptor (functioning as a competitive inhibitor of
the protein of the present invention). Partial polypeptides of the
present invention can be produced by genetic engineering
techniques, known peptide synthesis methods, or by cleaving the
protein of the present invention with a suitable peptidase.
[0042] Moreover, the present invention relates to DNA encoding the
protein of the present invention. In addition to being used for the
production of the protein of the present invention either in vivo
or in vitro as previously mentioned, the DNA of the present
invention may also be applied in, for example, gene therapy against
diseases caused by aberrations of the gene encoding the protein of
the present invention. Any type of DNA, such as cDNA synthesized
from mRNA, genomic DNA, or synthetic DNA, can be used, so long as
the DNA encodes the protein of the present invention. Also as long
as they can encode the present protein, DNAs comprising arbitrary
sequences based on the degeneracy of the genetic code are also
included.
[0043] The DNA of the present invention can be prepared by using
methods known in the art. For example, a cDNA library can be
constructed from cells expressing the protein of the present
invention and hybridization can be conducted using a part of the
DNA sequence of the present invention (for example, DNA sequence
shown in SEQ ID NO:1) as a probe. Alternatively, the DNA of the
present invention can be obtained by preparing RNA from cells
expressing the protein of the present invention, synthesizing
oligo-DNAs based on the DNA sequence of the present invention (for
example, the DNA sequence shown in SEQ ID NO:1), and amplifying the
cDNA encoding the protein of the present invention by PCR using the
oligonucleotides as primers.
[0044] The nucleotide sequence of the obtained cDNA is determined
to find an open reading frame, and thereby the amino acid sequence
of the protein of the invention can be obtained. The cDNA obtained
may also be used as a probe for screening a genomic library to
isolate genomic DNA.
[0045] More specifically, mRNAs may first be prepared from a cell,
tissue, or organ (e.g., organs such as the lungs, liver, kidney,
etc. or from an embryo) in which the protein of the invention is
expressed. Known methods can be used to isolate mRNAs; for
instance, total RNA is prepared by guanidine ultracentrifugation
(Chirgwin et al., Biochemistry 18:5294-5299, 1979) or AGPC method
(Chomczynski et al., Anal. Biochem. 162:156-159, 1987), and mRNA is
purified from total RNA using an mRNA Purification Kit (Pharmacia),
and such. Alternatively, mRNA may be directly purified by the
QUICKPREP.TM. mRNA Purification Kit (Pharmacia).
[0046] The obtained mRNA is used to synthesize cDNA using reverse
transcriptase. cDNA may be synthesized by using a kit such as the
AMV Reverse Transcriptase First-strand cDNA Synthesis Kit
(Seikagaku Kogyo). Alternatively, cDNA may be synthesized and
amplified 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), using the synthesized DNA as a primer,
the 5'-AMPLI FINDER RACE.TM. Kit (Clontech), and polymerase chain
reaction (PCR).
[0047] A desired DNA fragment is prepared from the PCR products and
ligated with a vector DNA. The recombinant vectors are used to
transform E. coli and such, and a desired recombinant vector is
prepared from a selected colony. The nucleotide sequence of the
desired DNA may be verified by conventional methods, such as
dideoxynucleotide chain termination.
[0048] The DNA of the invention may be designed to have a
nucleotide sequence having a high expression efficiency by taking
into account the frequency of codon usage in the host used for the
expression (Grantham et al., Nucleic Acids Res. 9:43-74, 1981). The
DNA of the present invention may be altered by a commercially
available kit or a conventional method. For instance, the DNA may
be altered by digestion with restriction enzymes, insertion of a
synthetic oligonucleotide or an appropriate DNA fragment, addition
of a linker, or insertion of an initiation codon (ATG) and/or stop
codon (TAA, TGA, or TAG).
[0049] Specifically, the DNA of the present invention includes a
DNA comprising nucleotides from nucleotide A at position 87 to
nucleotide T at position 752 of SEQ ID NO:1, and a DNA comprising
nucleotides from nucleotide T at position 159 to nucleotide T at
position 752 in the nucleotide sequence of SEQ ID NO:1.
[0050] The DNA of the present invention also includes a DNA that
hybridizes to a DNA comprising the nucleotide sequence indicated in
SEQ ID NO:1 under stringent conditions and is functionally
equivalent to the protein described in SEQ ID NO:2. Examples of
hybridization conditions include the conditions previously
described. The hybridized DNA may preferably be naturally occurring
DNA, such as cDNA or chromosomal DNA.
[0051] The DNA of the present invention can be used to produce the
protein of the present invention as a recombinant protein. In
addition, when there is a defect in the DNA encoding the protein of
the present invention, the DNA of the present invention may also be
applied to functional inhibition by an antisense, or gene therapy
by substituting it with the normal gene.
[0052] 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.
[0053] 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. 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. 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, 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, the comparison is made to a segment of the reference
sequence of the same length (excluding any loop required by the
homology calculation).
[0054] As used herein, "% identity" of two amino acid sequences, or
of two nucleic acid sequences, is determined using the algorithm of
Karlin and Altschul (PNAS USA 87:2264-2268, 1990), modified as in
Karlin and Altschul, PNAS 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.
[0055] The present invention also relates to a vector into which
the DNA of the present invention is inserted. The vectors of the
present invention are useful for carrying the DNA of the present
invention and to express the protein of the present invention in a
host cell.
[0056] When E. coli is used as the host cell, any vector can be
used as long as it comprises an "ori", to amplify and mass-produce
the vector in E. coli (e.g., JM109, DH5.alpha., HB101, or XL1
Blue), and a marker gene for selecting the transformed E. coli
(e.g., a drug-resistant gene selected by a drug (e.g., ampicillin,
tetracycline, kanamycin, or chloramphenicol). For example,
M13-series vectors, pUC-series vectors, pBR322, pBluescript,
pCR-Script, and such, can be used. Other than the vectors used
above, pGEM-T, pDIRECT, pT7, and so on, can also be used for
subcloning and excision of the cDNA. When using a vector to produce
the protein of the present invention, an expression vector is
especially useful. When, for example, the objective is to be
expressed in E. coli, the expression vector should have the above
characteristics in order to be amplified in E. coli. When E. coli,
such as JM109, DH5a, HB101, or XL1 Blue, are used as the host cell,
the vector should have a promoter, for example, lacZ promoter (Ward
et al., Nature 341:544-546, 1989; FASEB J. 6:2422-2427, 1992), araB
promoter (Better et al., Science 240:1041-1043, 1988), or T7
promoter, that can efficiently promote the expression of the
desired gene in E. coli. Other examples of the vectors are
pGEX-5X-1 (Pharmacia), QIAEXPRESS.RTM. system (Qiagen), pEGFP, and
pET (for this vector, BL21, a strain expressing T7 RNA polymerase,
is preferably used as the host).
[0057] In addition, a signal sequence for secreting a polypeptide
may be contained in the vector. The pelB signal sequence (Lei et
al., J. Bacteriol. 169:4379, 1987) can be used as the signal
sequence for protein secretion when the secretory protein is
produced into the periplasm of E. Coli. Introduction of the vector
into host cells can be carried out using, for example, the calcium
chloride method, electroporation, and so forth.
[0058] Examples of vectors used for producing the protein of the
invention other than those derived from E. Coli include, for
example, expression vectors derived from mammals (e.g., pcDNA3
(Invitrogen), pEGF-BOS (Nucleic Acids. Res. 18(17):5322, 1990),
pEF, and pCDM8), those derived from insect cells (e.g., the
"Bac-to-BAC baculovirus expression system" (GIBCO BRL) and
pBacPAK8), those derived from plants (e.g., pMH1 and pMH2), those
derived from animal viruses (e.g., pHSV, pMV, and pAdexLcw), those
derived from retroviruses (e.g., pZIpneo), those derived from yeast
(e.g., "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01),
and those derived from Bacillus subtilis (e.g., pPL608 and
pKTH50).
[0059] For expression in animal cells such as CHO cells, COS cells,
or NIH3T3 cells, it is essential for the vector to have a promoter
necessary for expression in the cells, such as the SV40 promoter
(Mulligan et al., Nature 277:108, 1979), MMLV-LTR promoter,
EF1.alpha. promoter (Mizushima et al., Nucleic Acids Res. 18:5322,
1990), and CMV promoter. More preferably, such a vector comprises a
gene for selecting cell transformation (for example, a
drug-resistant gene that allows selection by a drug (such as
neomycin or G418). Examples of vectors having such characteristics
include, for example, pMAM, pDR2, pBK-RSV, PBK-CMV, pOPRSV, and
pOP13.
[0060] Moreover, for stably expressing a gene and amplifying the
number of copies of the gene within cells, one example of a method
that can be used is the method in which a vector (such as pCH01)
having a DHFR gene that compensates for a defect in the nucleic
acid synthesis pathway is inserted into CHO cells, which is
followed by amplification by methotrexate (MTX). For transient gene
expression, an example method is one in which COS cells, having a
gene on their chromosomes that expresses SV40 T antigen, are
transformed with a vector (such as pcD) having an SV40 replication
origin. Replication origins derived from polioma virus, adenovirus,
bovine papillomavirus (BPV), and so forth can be also used.
Moreover, in order to amplify the number of gene copies in a host
cell line, the expression vector can contain aminoglycoside
transferase (APH) gene, thymidine kinase (TK) gene, E. coli
xanthine guanine phosphoribosyl transferase (Ecogpt) gene,
dihydrofolic acid reductase (dhfr) gene, and so forth, as a
selective marker.
[0061] On the other hand, for expressing the DNA of the present
invention in the living body of animals, a method in which the DNA
is first incorporated into a suitable vector, and then the vector
is introduced into the living body by the retrovirus method,
liposome method, cationic liposome method, adenovirus method, and
so forth, may be used. Thereby, gene therapy can be carried out
against diseases caused by a mutation in the gene that encodes the
protein of the present invention. For example, without limitation,
adenovirus vectors (e.g., pAdexlcw) and retrovirus vectors (e.g.,
pZIPneo) are used as vectors. General gene manipulation techniques
such as the insertion of the DNA of the present invention into a
vector can be carried out in accordance with ordinary methods
(Molecular Cloning, 5.61-5.63). Administration into the living body
may be carried out by either the ex vivo method or in vivo
method.
[0062] Furthermore, the present invention relates to a host cell
into which the vector of the present invention has been introduced.
There are no particular restrictions on the host cell, and
includes, for example, E. coli and various animal cells. The host
cell of the present invention can be used, for example, as a
production system for the production and expression of the protein
of the present invention. Production systems for producing the
protein include both in vitro and in vivo systems. Examples of in
vitro production systems include those using eukaryotic cells or
prokaryotic cells.
[0063] When using eukaryotic cells, for example, animal cells,
plant cells, and fungal cells can be used as the host. Known
examples of animal cells include mammalian cells such as CHO (J.
Exp. Med. 108:945, 1995), COS, 3T3, myeloma, BHK (baby hamster
kidney), HeLa, and Vero, amphibian cells such as Xenopus oocytes
(Valle et al., Nature 291:358-340, 1981), and insect cells such as
sf9, sf21, and Tn5. Particularly preferable CHO cells are those
deficient in the DHFR gene, dhfr-CHO (Proc. Natl. Acad. Sci. USA
77:4216-4220, 1980) and CHO K-1 (Proc. Natl. Acad. Sci. USA
60:1275, 1968). For mass expression in animal cells, CHO cells are
particularly preferable. A vector can be introduced into host cells
by, for example, the calcium phosphate method, DEAE dextran method,
method using cationic ribosome DOTAP (Boehringer-Mannheim),
electroporation, and lipofection.
[0064] Known examples of plant cells used as protein production
systems include cells derived from Nicotiana tabacum, and these
cells can be cultured as a callus culture. Yeasts such as
Saccharomyces species, e.g., Saccharomyces cerevisiae, as well as
filamentous bacteria such as Aspergillus species, e.g., Aspergillus
niger are known as fungal cells.
[0065] For prokaryotic cells, bacterial cells can be used as the
production system. Examples of bacterial cells include E. coli such
as E. coli JM109, DH5a and HB101, as well as Bacillus subtilis.
[0066] These cells are transformed by desired DNA, and the
resulting transformants are cultured in vitro to obtain the
protein. Transformants can be cultured using known methods. Culture
medium such as DMEM, MEM, RPMI1640, or IMDM may be used for animal
cells with or without serum supplements such as fetal calf serum
(FCS). The pH of the culture medium is preferably between about 6
and 8. Such cells are typically cultured at about 30 to 40.degree.
C. for about 15 to 200 hr, and the culture medium may be replaced,
aerated, or stirred as necessary.
[0067] Animal and plant hosts may be used for in vivo
protein-producing systems. For example, a desired DNA can be
introduced into an animal or plant host. Encoded proteins are
produced in vivo, and then recovered. These animal and plant hosts
are included in the host cells of the present invention.
[0068] Animals to be used for the production systems described
above include, but are not limited to, mammals and insects. Mammals
such as goats, pigs, sheep, mice, and cattle may be used (Vicki
Glaser, SPECTRUM Biotechnology Applications, 1993). Alternatively,
the mammals may be transgenic animals.
[0069] For instance, a desired DNA may be prepared as a fusion gene
by fusing it with a gene that encodes a protein specifically
produced into milk, such as goat .beta. casein gene. DNA fragments
comprising the fusion gene are injected into goat embryos, which
are then introduced back into female goats. Proteins of interest
can be recovered from milk produced by the transgenic goats (i.e.,
those born from the goats that had received the modified embryos)
or from their offspring. To increase the amount of milk containing
the proteins produced by transgenic goats, appropriate hormones may
be administered (Ebert et al., Bio/Technology 12:699-702,
1994).
[0070] Alternatively, insects, such as the silkworm, may be used. A
DNA encoding the desired protein inserted into a baculovirus can be
used to infect silkworms, and the desired protein can be recovered
from their body fluids (Susumu et al., Nature 315:592-594,
1985).
[0071] As plants, for example, tobacco can be used. When using
tobacco, DNA encoding the desired protein may be inserted into a
plant expression vector, such as pMON530, which is introduced into
bacteria, such as Agrobacterium tumefaciens. Then the bacteria is
used to infect tobacco, such as Nicotiana tabacum, and a desired
polypeptide is recovered from their leaves (Julian et al., Eur. J.
Immunol. 24:131-138, 1994).
[0072] A protein of the present invention obtained as above may be
isolated from the inside or outside (such as the culture medium) of
the host cell, and purified as a substantially pure homogeneous
protein. The method for protein isolation and purification is not
limited to any specific method; in fact, any standard method may be
used. For instance, column chromatography, filters,
ultrafiltration, salt precipitation, solvent precipitation, solvent
extraction, distillation, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, isoelectric point electrophoresis, dialysis,
and recrystallization may be appropriately selected and combined to
isolate and purify the protein.
[0073] For chromatography, for example, affinity chromatography,
ion-exchange chromatography, hydrophobic chromatography, gel
filtration, reverse phase chromatography, adsorption
chromatography, and such may be used (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed.
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1996). These chromatographies may be performed by liquid
chromatography such as HPLC and FPLC. Thus, the present invention
provides highly purified proteins produced by the above
methods.
[0074] A protein of the present invention may be arbitrarily
modified or peptides may be partially deleted by treating it with
an appropriate protein modification enzyme before or after
purification. Useful protein modification enzymes include, but are
not limited to, trypsin, chymotrypsin, lysylendopeptidase, protein
kinase, glucosidase and such.
[0075] The present invention also relates to an antibody that binds
to the protein of the invention. An antibody of the invention may
take any form, including monoclonal antibodies, as well as
polyclonal antibodies. Furthermore, antiserum obtained by
immunizing an animal such as a rabbit, or the like, with the
protein of the invention, all classes of polyclonal and monoclonal
antibodies, human antibodies, and humanized antibodies produced by
genetic recombination are included.
[0076] A protein of the invention used as an antigen to obtain an
antibody may be derived from any animal species, but preferably it
is from a mammal such as a human, mouse, or rat, more preferably
from a human. A human-derived protein may be obtained from the
nucleotide or amino acid sequences disclosed herein.
[0077] A whole protein or a partial peptide of a protein may be
used as a sensitizing antigen in the present invention. A partial
peptide may be, for example, an amino (N)-terminal or carboxy
(C)-terminal fragment of a protein. Herein, an "antibody" is
defined as an antibody that specifically reacts with either the
full length or a fragment of a protein.
[0078] A gene encoding the protein of the invention or a its
fragment may be inserted into a known expression vector, a host
cell as described herein may be transformed with the vector, and
the desired protein or its fragment for use as a sensitizing
antigen may be obtained from the outside or inside of host cells by
any standard method. Alternatively, cells expressing the protein or
their lysates, or a chemically synthesized protein may be used as
the antigen.
[0079] Any mammal may be immunized with the antigen, but preferably
the compatibility with parental cells used for cell fusion is taken
into account. In general, animals of Rodentia, Lagomorpha, or
Primates are used.
[0080] Animals of Rodentia include, for example, mice, rats, and
hamsters. Animals of Lagomorpha include, for example, rabbits.
Animals of Primates include, for example, monkeys of Catarrhini
(old world monkey) such as Macaca fascicularis, rhesus monkeys,
sacred baboons, or chimpanzees.
[0081] Methods for immunizing animals with antigens are known in
the art. In a standard method, a sensitizing antigen is injected
intraperitoneally or subcutaneously to mammals. More specifically,
an appropriate amount of a standard adjuvant, such as Freund's
complete adjuvant, is mixed with the sensitizing antigen, diluted
and suspended in an appropriate amount of phosphate buffered saline
(PBS), physiological saline, or such, emulsified, and then
administered to mammals. Preferably, this is followed by several
administrations of antigen mixed with an appropriately amount of
Freund's incomplete adjuvant every 4 to 21 days. An appropriate
carrier may also be used for immunization of sensitizing antigens.
After an immunization as above, the serum is examined for an
increase of the amount of desired antibodies by a standard
method.
[0082] Polyclonal antibodies against the protein of the present
invention may be prepared by collecting blood from the immunized
mammal examined for an increase of desired antibodies in the serum,
and by separating serum from the blood by any conventional method.
Polyclonal antibodies may be used as serum containing polyclonal
antibodies, or if necessary, a fraction containing the polyclonal
antibodies may be isolated from the serum for use. For example,
immunoglobulin G or M can be prepared by using an affinity column
coupled with the protein of the present invention to obtain a
fraction that recognizes only the protein, followed by purifying
this fraction using a protein A column or protein G column.
[0083] To prepare a monoclonal antibody, immune cells are collected
from the mammal sensitized with the above antigen after verifying
that the desired antibody level has increased in the serum. The
cells are then subjected to cell fusion. The immune cells used for
cell fusion are preferably obtained from spleen. The other parent
cell that is fused with the above immune cell is preferably a
mammalian myeloma cell, and more preferably a myeloma cell that has
acquired a special feature that can be used for the selection of
fusion cells by a drug.
[0084] Cell fusion of the above immune cell and myeloma cell may be
performed by any standard method, such as those described in
literature (Galfre et al., Methods Enzymol. 73:3-46, 1981).
[0085] Resulting hybridomas obtained by cell fusion may be selected
by cultivating them in a standard selection medium, such as HAT
medium (hypoxanthine, aminopterin, and thymidine containing
medium). The cell culture is typically continued in the HAT medium
until all cells other than the desired hybridoma (non-fused cells)
die, usually from several days to several weeks. Then, the standard
limiting dilution is performed to screen and clone a hybridoma cell
producing the desired antibody.
[0086] Besides the above method in which a nonhuman animal is
immunized with an antigen for preparing hybridoma, human
lymphocytes such as those infected by the EB virus may be immunized
with a protein, protein expressing cells, or their lysates in
vitro. Then, the immunized lymphocytes are fused with human-derived
myeloma cells capable of indefinite division, such as U266, to
yield a hybridoma producing a desired human antibody capable of
binding to the protein (Unexamined Published Japanese Patent
Application (JP-A) No. Sho 63-17688).
[0087] Subsequently, the hybridomas thus obtained are transplanted
into the abdominal cavity of a mouse from which the ascites is
collected. The monoclonal antibodies thus obtained can be purified
by, for example, ammonium sulfate precipitation or by column
chromatography using a protein A or protein G column, a DEAE ion
exchange column, an affinity column, and such to which the protein
of the invention is coupled. The antibody of the invention can be
used not only for purifying and detecting a protein of the
invention, but also as a candidate for an agonist or antagonist to
a protein of the present invention. The antibody is also expected
to be used in antibody therapy against diseases related to the
present protein. When using the resulting antibody for the purpose
of administration to the human body (antibody therapy), human
antibodies or humanized antibodies are preferred to reduce
immunogenicity.
[0088] For example, human antibodies against a protein can be
obtained using hybridomas obtained by fusing myelomas with
antibody-producing cells, which are obtained by immunizing
transgenic animals having the human antibody gene repertoire with
an antigenic protein, cells expressing the protein, or a lysate
thereof (See WO92-03918, WO93-2227, WO94-02602, WO94-25585,
WO96-33735 and WO96-34096).
[0089] Alternatively, an immune cell, such as an immunized
lymphocyte, which produces antibodies may be immortalized by an
oncogene and used for preparing monoclonal antibodies.
[0090] Monoclonal antibodies thus obtained can be also be
recombinantly prepared using conventional genetic engineering
techniques (see, for example, Borrebaeck C. A. K. and Larrick J. W.
Therapeutic Monoclonal Antibodies, published in the United Kingdom
by MacMillan Publishers LTD, 1990). A recombinant antibody can be
produced by cloning a DNA encoding the antibody from an immune cell
such as a hybridoma or an immunized lymphocyte producing the
antibody, inserting this DNA into an appropriate vector, and
introducing this into a host cell. The present invention also
provides recombinant antibodies prepared as described above.
[0091] An antibody of the present invention may be a fragment of an
antibody or a modified antibody, so long as it binds to one or more
of the proteins of the invention. For instance, the antibody
fragment may be Fab, F (ab').sub.2, Fv, or single chain Fv (scFv),
in which Fv fragments from H and L chains are ligated by an
appropriate linker (Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883, 1988). More specifically, an antibody fragment may be
generated by treating an antibody with an enzyme such as papain or
pepsin. Alternatively, a gene encoding the antibody fragment may be
constructed, inserted into an expression vector, and expressed in
an appropriate host cell (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).
[0092] An antibody may be modified by conjugation with a variety of
molecules, such as polyethylene glycol (PEG). The present invention
encompasses such modified antibodies. A modified antibody can be
obtained by chemically modifying an antibody. These modification
methods are conventional in the field.
[0093] An antibody of the present invention may be obtained as a
chimeric antibody comprising a variable region derived from a
nonhuman antibody and the constant region derived from a human
antibody. Alternatively, the present antibody may be obtained as a
humanized antibody, comprising the complementarity-determining
region (CDR) derived from a nonhuman antibody, the framework region
(FR) constant region derived from a human antibody. Such antibodies
can be prepared by using known techniques.
[0094] An Antibody obtained as above may be purified to
homogeneity. Methods generally used for separating and purifying
ordinary proteins may be used for separating and purifying the
present antibody. For example, the antibody can be separated and/or
purified by the appropriate selection and combined use of column
chromatographies such as affinity chromatography and the like,
filters, ultrafiltration, salting-out, dialysis, SDS polyacrylamide
gel electrophoresis, isoelectric focusing, etc. (Antibodies: A
Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor
Laboratory, 1988), without limitation. The concentration of the
antibody obtained as above may be determined by measuring the
absorbance or by an enzyme-linked immunosorbent assay (ELISA), and
such.
[0095] A column used in affinity chromatography is exemplified by
protein A column or protein G column. For example, protein A column
includes HYPER D.TM., POROS.TM., and SEPHAROSE.TM. F. F.
(Pharmacia).
[0096] In addition to affinity chromatography, the chromatography
method includes, for example, ion-exchange chromatography,
hydrophobic chromatography, gel filtration, reverse-phase
chromatography, adsorption chromatography, and the like (Strategies
for Protein Purification and Characterization: A Laboratory Course
Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory
Press, 1996). The chromatographies can be carried out by
liquid-phase chromatography such as HPLC, FPLC, or the like.
[0097] For example, the determination of absorbance, Enzyme-linked
immunosorbent assay (ELISA), enzyme immunoassay (EIA),
radioimmunoassay (RIA), and/or immunofluorescence may be used to
measure the antigen binding activity of the antibody of the
invention. In ELISA, the antibody of the present invention is
immobilized on a plate, protein of the invention is applied to the
plate, and then a sample containing a desired antibody, such as
culture supernatant of antibody producing cells or purified
antibodies, is applied. Then, a secondary antibody, which
recognizes the primary antibody and which is labeled with an enzyme
such as alkaline phosphatase, is applied, and the plate is
incubated. After washing, an enzyme substrate, such as
p-nitrophenyl phosphate, is added to the plate, and the absorbance
is measured to evaluate the antigen binding activity of the sample.
A fragment of the protein, such as a C-terminal or N-terminal
fragment, may also be used. BIACORE.RTM. (Pharmacia) may be used to
evaluate the activity of the antibody according to the present
invention.
[0098] The above methods allow the detection or measurement of the
protein of the invention, by exposing the antibody of the invention
to a sample presumed to contain the protein of the invention, and
detecting or measuring the immune complex formed by the antibody
and the protein.
[0099] Because the method of detection or measurement of the
protein according to the invention can specifically detect or
measure a protein, the method may be useful in a variety of
experiments in which the protein is used.
[0100] The present invention also relates to a nucleotide
comprising at least 15 nucleotides that hybridizes to the DNA (SEQ
ID NO:1) encoding the protein described in SEQ ID NO:2 or to a
complementary strand thereof. Nucleotides of the present invention
specifically hybridize to DNA (SEQ ID NO:1) encoding the protein
described in SEQ ID NO:2 or to a complementary strand thereof.
Here, the term "specifically hybridize" refers to the absence of
significant cross-hybridization with DNA encoding other proteins
under normal hybridization conditions, and preferably under
stringent hybridization conditions. Such nucleotides include
probes, primers, nucleotides, and nucleotide derivatives (e.g., DNA
encoding antisense oligonucleotides and ribozyme) that are able to
specifically hybridize to DNA encoding the protein of the present
invention or to complementary DNA thereof. In addition, such
nucleotides can also be used for the production of DNA chips.
[0101] The present invention comprises, for example, an antisense
oligonucleotide that hybridizes to any part of the nucleotide
sequence of SEQ ID NO:1. The antisense oligonucleotide is
preferably an antisense of a continuous sequence comprising at
least 15 nucleotides or more within the nucleotide sequence SEQ ID
NO:1. More preferably, the above continuous sequence comprising at
least 15 nucleotides or more that contains a translation initiation
codon.
[0102] A derivative or modified form of an antisense
oligonucleotide may also be used. The latter form may be modified
with lower alkylphosphonate such as methylphosphonate or
ethylphosphonate, or with phosphorothioate, or
phosphoroamidate.
[0103] Herein, an antisense oligonucleotide is not restricted to
one in which all nucleotides are complementary to the corresponding
nucleotides within a given region of a DNA or mRNA, as long as it
can specifically hybridize to the nucleotide sequence of SEQ ID
NO:1, it may have one or more nucleotide mismatches.
[0104] Such nucleotides have a homology of at least 70%, preferably
80% or more, more preferably 90% or more, and even more preferably
95% or more within a continuous sequence comprising at least 15
nucleotides or more. To determine the degree of homology, the
algorithm described herein may be used. As described in the
following Examples, the above DNA is useful as a probe for
detecting or isolating a DNA encoding the protein of the invention,
or as a primer for its amplification.
[0105] A derivative of an antisense oligonucleotide of a present
invention may act on cells producing the protein of the invention
and bind to a DNA or mRNA encoding the protein, and then, it may
inhibit the expression of the protein of the invention by
inhibiting its transcription or translation, or by promoting the
degradation of mRNA, and thereby inhibiting the function of the
protein of the invention.
[0106] A derivative of an antisense oligonucleotide of the present
invention may be mixed with an appropriate nucleotide that is
inactive against the derivative, and used as a medicine for
external application, such as an ointment or poultice.
[0107] If necessary, it may be mixed with an excipient, isotonizing
agent, solubilizing agent, stabilizer, preservative, pain-killer,
or the like, and prepared as a tablet, powder, granule, capsule,
liposome capsule, injectable solution, liquid formulation, nose
drops, freeze-dried agent, etc. The above may be achieved according
to standard methods.
[0108] For treating patients, a derivative of an antisense
oligonucleotide of the present invention may be, for example,
directly applied to the affected area of a patient, or administered
into blood vessels so as to finally reach the affected area.
Moreover, the derivative may be encapsulated in
antisense-encapsulating materials such as liposomes, poly-L-lysine,
lipid, cholesterol, lipofectin, or their derivatives in order to
increase durability and/or membrane permeability.
[0109] Dose of the derivative of the antisense oligonucleotide of
the present invention may be appropriately adjusted depending on
the patient's condition, and an appropriate amount such as 0.1 to
100 mg/kg, or more preferably 0.1 to 50 mg/kg may be
administered.
[0110] As an antisense oligonucleotide of the present invention
inhibits expression of the protein of the invention, it can be
utilized as an inhibitor of a biological activity of the protein of
the invention. An inhibitor of expression comprising an antisense
oligonucleotide of the present invention is useful because it can
inhibit the a biological activity of the protein of the
invention.
[0111] Further, the present invention relates to a method for
screening compounds that bind to the protein of the present
invention using the protein, as well as compounds which can be
isolated by the screening method (e.g., receptors, agonists, and
antagonists).
[0112] The protein of the present invention used for screening may
be a recombinant protein, natural protein, or a partial peptide.
One embodiment of this screening method comprises the steps of (a)
contacting a test sample with the protein of the present invention
or its partial peptide, and (b) selecting a compound having an
activity for binding to the protein or its partial peptide. Without
limitation, the test sample includes cell extracts, cell culture
supernatants, products of fermentation microorganisms, marine
organism extracts, plant extracts, purified or crude proteins,
peptides, non-peptide compounds, synthetic low molecular weight
compounds, and naturally-occurring compounds. The protein of the
present invention can be contacted with the test sample in the form
of, for example, a purified protein, solubilized protein, a protein
bound to a carrier, a fusion protein with another protein, a
protein expressed on a cell membrane, or as a membrane
fraction.
[0113] Numerous methods known to those skilled in the art can be
used as methods for screening a protein that binds to the protein
of the present invention using the protein. Such screenings can be
carried out, for example, by the immunoprecipitation method.
Specifically, the method can be carried out as follows. The gene
encoding the protein of this invention is expressed by inserting
the gene downstream of a promoter for expressing a foreign gene,
such as pSV2neo, pcDNA I, pCD8, etc., and expressing the gene in
animal cells, etc. Any generally used promoter 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), EF-1.alpha. promoter (Kim et al., Gene
91:217-223, 1990), CAG promoter (Niwa et al., Gene 108:193-200,
1991), RSV LTR promoter (Cullen, Methods in Enzymol. 152:684-704,
1987), SR.alpha. promoter (Takebe et al., Mol. Cell. Biol. 8:466,
1988), CMV immediate early promoter (Seed et al., Proc. Natl. Acad.
Sci. USA 84:3365-3369, 1987), SV40 late promoter (Gheysen et al.,
J. Mol. Appl. Genet. 1:385-394, 1982), Adenovirus late promoter
(Kaufman et al., Mol. Cell. Biol. 9:946, 1989), HSV TK promoter,
etc. Transfer of a foreign gene into animal cells for expression
can be performed by any one 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), etc. The protein of
this invention can be expressed as a fusion protein having a
recognition site for a monoclonal antibody by introducing such a
site the specificity of which has been established, into the N- or
C-terminal of the protein of this invention. For this purpose, a
commercial epitope-antibody system can be utilized (Jikken Igaku,
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 fluorescence protein (GFP), and such, via a
multi-cloning site.
[0114] To minimize the alteration in properties of the protein of
this invention due to fusion protein formation, a method for
preparing a fusion protein by introducing only a small epitope
portion comprising several to ten amino acid residues has been
reported. For example, the epitopes of polyhistidine (His-tag),
influenza hemagglutinin (HA), human c-myc, FLAG, Vesicular
stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein
(T7-tag), human herpes simplex virus glycoprotein (HSV-tag), E-tag
(epitope on the monoclonal phage), and such, and monoclonal
antibodies that recognize these epitopes can be utilized as
epitope-antibody systems for screening proteins binding to the
protein of this invention (Jikken Igaku, Exp. Med. 13:85-90,
1995).
[0115] In immunoprecipitation, immune complexes are formed by
adding these antibodies to a cell lysate prepared using suitable
surfactants. This immune complex comprises the protein of this
invention, a protein capable of binding to the protein, and an
antibody. Immunoprecipitation can also be performed using an
antibody against the protein of this invention besides antibodies
to the above-described epitopes. An antibody to the protein of this
invention can be prepared by inserting a gene encoding the protein
of this invention into an appropriate expression vector of E. coli
to express it in the bacterium, purifying the protein thus
expressed, and immunizing rabbits, mice, rats, goats, chicken, and
such, with the purified protein. The antibody can also be prepared
by immunizing the above-described animals with a partial peptide of
the protein of this invention.
[0116] Immune complexes can be precipitated using, for example,
Protein A Sepharose and Protein G Sepharose when the antibody is a
murine IgG antibody. In addition, in the case where the protein of
this invention is prepared as a fusion protein with an epitope of,
for example, GST, and such, the immune complex can be formed using
a substance that specifically binds to this epitope, such as
glutathione-Sepharose 4B, and such, giving the same result as in
the case where the antibody for the protein of this invention is
used.
[0117] Immunoprecipitation, in general, may be carried out
according to, or following the method described in literature
(Harlow et al.: Antibodies, pp. 511-552, Cold Spring Harbor
Laboratory publications, New York, 1988).
[0118] SDS-PAGE is generally used for the analysis of
immunoprecipitated proteins. Bound proteins can be analyzed based
on the molecular weights of proteins using a gel of an appropriate
concentration. In this case, although proteins bound to the protein
of this 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 medium containing radio isotope-labeled
.sup.35S-methionine and .sup.35S-cysteine 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.
[0119] In addition, for example, West Western blotting method
(Skolnik et al., Cell 65:83-90, 1991) can be used to isolate a
protein that binds to the protein of the present invention using
the protein. Namely, a cDNA library is prepared using a phage
vector (such as .lamda.gt11 or ZAP) from cells, tissue, or organs
(such as the heart, lung, liver, kidney, and so forth, and embryos)
in which a protein that binds to the protein of the present
invention is presumed to be expressed, and then, this cDNA library
is expressed on LB-agarose. The expressed protein is fixed on a
filter and the fixed protein is purified, the labeled protein of
the present invention is reacted with the above filter, and plagues
that express a protein bound to the protein of the present
invention is detected using the label. Methods for labeling a
protein of the present invention include those that use the binding
properties of biotin and avidin, those that use antibodies that
specifically bind to the protein of the present invention or a
peptide or polypeptide (such as GST) that has been fused with the
protein, those that use radioisotopes or fluorescence.
[0120] In addition, other embodiments of the screening method of
the present invention include methods that use a two-hybrid system
using cells (such as the "MATCHMAKER.TM. Two-Hybrid System",
"Mammalian MATCHMAKER.TM. Two-Hybrid Assay Kit", and
"MATCHMAKER.TM. One-Hybrid System" (all by Clontech), the
"HYBRIZAP.TM. Two-Hybrid Vector System" (Stratagene), and the "CYTO
TRAP.TM. two-hybrid system" (Stratagene); References: Dalton et
al., Cell 68:597-612, 1992; Fields et al., Trends. Genet.
10:286-292, 1994).
[0121] In a two-hybrid system, a cDNA library is prepared from
cells in which protein that bind to the protein of the present
invention is presumed to be expressed by expressing the protein of
the present invention in yeast cells by fusing it with the SRF DNA
binding region or GAL4 DNA binding region, and expressing in a form
that fuses with the VP 16 or GAL4 transcriptional activation
region. This cDNA library is then introduced into the above yeast
cells, and library-derived cDNA is isolated from the positive
clones detected (when a protein that binds to the protein of the
present invention is expressed in yeast cells, a reporter gene is
activated by their binding, thereby making it possible to confirm
the positive clones). Protein corresponding to the isolated cDNA
can then be obtained by introducing said cDNA into E. coli and
expressing it therein.
[0122] Examples of reporter genes that can be used include HIS3
gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene and PAI-1
(plasminogen activator inhibitor type 1) gene.
[0123] Screening of compounds that bind to the protein of the
present invention can be carried out using affinity chromatography.
For example, the protein of the present invention is immobilized on
a carrier of an affinity column, and then a test sample presumed to
express a protein that binds to the protein of the present
invention is applied to the column. In this case, for example, cell
extracts and cell lysates can be used as test samples. After
applying the test sample, the protein that binds to the protein of
the present invention can be prepared by washing the column.
[0124] DNA encoding the resulting protein can be obtained by
analyzing the amino acid sequence of the protein, synthesizing
oligo DNA based on that sequence, and screening a cDNA library by
using the DNA as a probe.
[0125] In the present invention, a biosensor utilizing the surface
plasmon resonance phenomena can be used as means for detecting or
determining bound compounds. Biosensors using surface plasmon
resonance phenomena allow real-time observation of the interaction
between the protein of the present invention and a test compound as
a surface plasmon resonance signal using a trace amount of the
protein and without labeling (for example, BIACORE.RTM.
(Pharmacia)). Thus, using a BIACORE.RTM. or other biosensor allows
one to evaluate the binding between the protein of the present
invention and a test compound.
[0126] In addition, examples of methods known to persons skilled in
the art for isolating compounds (including agonists and
antagonists) that bind to the protein of the present invention
without being limited to proteins, include: a method for screening
molecules that bind to the protein of the present invention by
allowing a synthetic compound, natural substance bank, or random
phage peptide display library to act on an immobilized protein of
the present invention; and a high-throughput screening method using
combinatorial chemistry technology (Wrighton et al., Science
273:458-464, 1996; Verdine, Nature 384:11-13, 1996; Hogan, Jr.,
Nature 384:17-19, 1996).
[0127] Compounds that can be isolated by the screening can be drug
candidates for promoting or inhibiting the activity of the protein
of the present invention, and can be applied to the treatment of
diseases caused by expression or functional abnormalities of the
protein of the invention. A substance in which a portion of the
structure of a compound, which has an activity for binding to the
protein of the present invention and which was obtained by using
the screening method of the present invention, is altered by
addition, deletion, and/or substitution is also included in the
compounds obtained by using the screening method of the present
invention.
[0128] When using the compound obtained by the screening method of
this invention and present protein as a drug for humans and
mammals, for example, mice, rats, guinea pigs, rabbits, chicken,
cats, dogs, sheep, pigs, cattle, monkeys, sacred baboons, and
chimpanzees, the isolated compound itself can be directly
administered to a patient, or it can be given after formulation by
using commonly known pharmaceutical preparation methods. For
example, according to the need, the drug can be taken orally as
sugarcoated tablets, capsules, elixirs, and microcapsules, or
parenterally in the form of injections of aseptic solutions or
suspensions with water or any other pharmaceutically acceptable
liquid. The compound may be formulated by mixing with, for example,
pharmacologically acceptable carriers or media, specifically,
sterilized water, physiological saline, plant oils, emulsifiers,
suspending agents, surfactants, stabilizers, flavoring agents,
excipients, vehicles, preservatives, binders, and so on, in a unit
dose form required for generally accepted drug implementation. The
amount of active ingredients in these preparations makes a suitable
dosage within the indicated range acquirable.
[0129] Examples for additives which can be mixed with tablets and
capsules are, binders such as gelatin, corn starch, tragacanth gum,
and arabic gum; excipients such as crystalline cellulose; swelling
agents such as corn starch, gelatin, and alginic acid; lubricators
such as magnesium stearate; sweeteners such as sucrose, lactose, or
saccharin; and 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 compositions for injections can be formulated
following normal drug implementations using vehicles such as
distilled water used for injections.
[0130] Physiological saline and isotonic liquids including glucose
or other 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, non-ionic surfactants, such as
polysorbate 80.TM. and HCO-50.
[0131] Sesame oil or soy-bean oil can be used as a oleaginous
liquid and may be used in conjunction with benzyl benzoate or
benzyl alcohol as a solubilizer; may be formulated with a buffer
such as phosphate buffer and sodium acetate buffer; a pain-killer
such as procaine hydrochloride; a stabilizer such as benzyl alcohol
and phenol; or an anti-oxidant. The prepared injection is filled
into a suitable ampule.
[0132] The administration to patients is done by methods commonly
known to those skilled in the art, such as by intra-arterial,
intravenous, or subcutaneous injections, and in addition, as
intranasal, bronchial, intramuscular, percutaneous, or oral
administrations. One skilled in the art can suitably select the
dosage according to the body-weight or age of a patient, or the
method of administration. Also, if the compound can be encoded by
DNA, the compound can be used for gene therapy by integrating the
DNA into a vector for gene therapy. Although the dosage amount and
method of administration differ according to the body-weight, age,
and symptoms of a patient, one skilled in the art can suitably
select these.
[0133] For example, although the dosage for a single administration
of the protein of the present invention varies depending on the
administration target, target organ, symptoms, administration
method, and so forth, the dosage in the form of, for example, an
injection preparation is usually considered to be in the range of
about 100 .mu.g to 20 mg per day for an adult (assuming the body
weight is 60 kg).
[0134] For example, although the dosage of a compound that binds to
the protein of the present invention or the dosage of a compound
that inhibits the activity of the protein of the present invention
varies according to the symptoms, in the case of oral
administration, the dosage for an adult (assuming the body weight
is 60 kg) is typically in the range of about 0.1 to 100 mg,
preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20
mg, per day.
[0135] In the case of parenteral administration, although the
dosage for a single administration also varies according to the
administration target, target organ, symptoms, and administration
method, the dosage in the form of, for example, an injection
preparation for an adult (assuming the body weight is 60 kg) is
usually in the range of about 0.01 to 30 mg, preferably about 0.1
to 20 mg, and more preferably about 0.1 to 10 mg, per day for a
convenient intravenous administration. In the case of other animals
as well, it is possible to administer an amount converted to 60 kg
of body weight or per area of body surface.
[0136] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0137] FIG. 1 shows the homology between the amino acid sequences
encoded by clone 106 (above; SEQ ID NO:2) and Drosophila twisted
gastrulation (TSG) gene (below; SEQ ID NO:5). Asterisks (*)
indicate identical amino acid sequences, while dots (.) indicate
similar amino acid sequences. Gaps are supplemented with bars.
[0138] All publications and patents cited herein are incorporated
by reference in their entirety.
DETAILED DESCRIPTION
[0139] The present invention is described below in detail using
examples, but it is not to be construed as being limited
thereto.
Example 1
Isolation of Clone 106
[0140] The AGM region was sampled from 11.5-day mice embryos, and
polyA(+) RNA was prepared using FAST TRACK.RTM. (Invitrogen).
Double-strand cDNA was synthesized using a random primer of the
SUPERSCRIPT.TM. Choice System (GIBCO BRL). BstXI adapter
(Invitrogen) was added after blunting the ends of the cDNA, and
then 400 bp or longer cDNA were fractionated using the SizeSep 400
Spun Column (Pharmacia).
[0141] After the cDNA was mixed with pMXGM(-)v-mpl.sup.M2 (see
Japanese Patent Application No. Hei 9-324912), which had been
treated with BstX1 (TAKARA) beforehand, it was ligated using T4 DNA
ligase. The resulting DNA was introduced into E. coli DH10B (GIBCO
BRL) by electroporation using GENE PULSER.RTM. (BioRad), and
cultured overnight. The cDNA library was purified using the
JETSTAR.TM. column (GENOMED).
[0142] Packaging cells BOSC23 (Proc. Natl. Acad. Sci. USA
90:8392-8396, 1993) were transfected with the cDNA library using
LIPOFECTAMINE.TM. (LIFE TECHNOLOGIES). BOSC23 were seeded into a
6-cm dish with DMEM (LIFE TECHNOLOGIES) containing 10% fetal calf
serum (FCS, JRH BIOSCIENCES), and then washed with DMEM 16 hours
later. 18 .mu.l of LIPOFECTAMINE.TM. diluted beforehand with 200
.mu.l of DMEM and 3 .mu.g of the cDNA library diluted with 200
.mu.l of DMEM were mixed together. This was kept standing at room
temperature for 15 minutes, then 1.6 ml of DMEM was added thereto,
and the mixture was added to the cells. After five hours, 2 ml of
DMEM containing 20% FCS was added to the mixture and cultured for
19 hours. Subsequently, the medium was replaced with 3 ml of DMEM
containing 10% FCS and the culture supernatant was collected 24
hours later. Mouse interleukin-3 (IL-3) and 10 .mu.g/ml of
hexadimethrine bromide were added to the culture supernatant
containing the recombinant virus, and Ba/F3 were suspended for
infection. 24 hours after the infection, the cells were washed
three times with PBS, and further cultured with RPMI1640 (LIFE
TECHNOLOGIES) containing 10% FCS. DNA was extracted from clones
that proliferated in the absence of IL-3 and amplified by PCR using
primers 5'-gggggTggACCATCCTCTA-3' (SEQ ID NO:3) and
5'-CgCgCAgCTgTAAACggTAg-3' (SEQ ID NO:4), designed to surround the
cDNA insertion site, followed by recovery of the cDNA fragment. PCR
was performed under the following conditions with the GENEAMP.RTM.
PCR System 2400 (Applied Biosystems) using 50 .mu.l of the reaction
mixture containing 500 ng of DNA, 500 .mu.M each of primer, 2.5
units of TAKARA LA.TM. Taq (TAKARA), 2.5 mM MgCl.sub.2, 0.3 mM
dNTPs, and enzyme-supplemented buffer: denaturing at 98.degree. C.
for 60 seconds, followed by 30 cycles of 98.degree. C. for 20
seconds, and 68.degree. C. for 120 seconds. The PCR reaction
product was electrophoresed on an agarose gel, the portion
containing the amplified fragment was excised, and then purified.
The nucleotide sequence of the resulting DNA fragment was
determined and translated to amino acids, then the isolated gene
(clone 106) was found to be 33% homologous at the amino acid level
with the Drosophila twisted gastrulation gene (TSG) (Mason et al.,
Genes and Develop. 8:1489-1501) (FIG. 1). Drosophila TSG gene is
thought to be one of the embryonic dorsal determining factors, and
the mutation of this gene prevents differentiation of only dorsal
midline cells derived from the mesoderm. This is considerably
different to the decapentaplegic (DP) gene, which is also a dorsal
determining factor considered to interact with TSG gene, where the
differentiation of the entire dorsal region is affected.
Example 2
Acquisition of Full-Length cDNA
[0143] A cDNA library of a 11.5 day-mouse embryo was synthesized in
the same manner as in Example 1 using an oligo dT primer and
screened using the cDNA fragment as the probe to obtain the
full-length cDNA. 2 .mu.g of the cDNA library was added to 50 .mu.l
of DH5.alpha. (GIBCO BRL) and left standing for 30 minutes on ice.
After applying heat shock for 30 seconds at 42.degree. C., the
mixture was allowed to stand for about 2 minutes on ice. After the
addition of 300 .mu.l of SOC, the mixture was cultured for 30
minutes at 37.degree. C. The mixture was then seeded into a 10-cm
dish LB plate (containing ampicillin) on which a NITROBIND.TM.
Nitrocellulose Transfer membrane (MICRON SEPARATIONS) was placed so
as to obtain 30,000-40,000 E. coli colonies per plate. The E. Coli
colonies that proliferated on the membrane were transferred to a
BIODYNE.RTM. A transfer membrane (Pall), and cultivated on the LB
plate for several hours. The BIODYNE.RTM. A transfer membrane was
then used for screening the cDNA library. After denaturing with a
denaturing solution (0.5 N NaOH and 0.5 M NaCl) for five minutes,
the membrane was neutralized with a neutralizing solution (0.5 M
Tris-HCl, pH 7.4 and 1.5 M NaCl). After gently washing with
2.times.SSC and drying up, the DNA and membrane were cross-linked
by irradiating with UV light at 1200 J.
[0144] Hybridization was performed according to the following
procedure. First, the membrane was pre-hybridized for 2 hours at
42.degree. C. in a hybridization buffer (50% formamide, 4.5%
Dextran Sulfate, 0.1 mg/ml of salmon sperm DNA, 6.times.SSC, and 1%
SDS). After labeling with RI using PRIME-IT.RTM. (Stratagene) and
after heat denaturing, 25 ng of clone 106 DNA to be used for the
probe was added to the hybridization buffer and left to stand
overnight.
[0145] The membrane was washed in two stages. First, the membrane
was washed for 10 minutes at 42.degree. C. with a washing buffer
(2.times.SSC and 0.1% SDS), and then for 30 minutes at 55.degree.
C. with a washing buffer (0.1.times.SSC and 0.1% SDS). The membrane
washed in this manner was then brought into close contact with an
X-ray film and developed by exposing to light at -80.degree. C.
[0146] One type of clone was obtained through the above procedure.
The clone, which was a 3986 bp cDNA, was found to have an open
reading frame (87-752) that encodes 222 amino acids, in which amino
acids 1 through 24 were presumed to be the signal sequence. The
nucleotide sequence of the cDNA is shown in SEQ ID NO:1, while the
encoded amino acid sequence is shown in SEQ ID NO:2.
Example 3
Expression Analysis of the cDNA Clone by Northern Hybridization
[0147] When Northern hybridization was performed using mouse
Multiple Tissue Northern Blot (Clontech) and the cDNA obtained in
Example 2 as the probe, signals of about 4.0 kb were found in the
heart, lung, liver, and kidney. These signals were also confirmed
to be expressed in 9, 10, 11, 12, and 13-day embryos.
INDUSTRIAL APPLICABILITY
[0148] The protein and gene discovered in the present invention
could be counterparts of Drosophila TSG gene in mice, which
suggests that they may be functionally similar. Through the
investigation of their roles in embryo development, the protein and
gene of the present invention may contribute to the elucidation of
mechanisms of differentiation and bone formation associated with
hematopoietic stem cell generation. In addition, they are also
useful as tools for developing therapeutic agents for the treatment
of diseases related to immune and hematopoiesis-systems and bone
formation.
Sequence CWU 1
1
513986DNAMus musculusCDS(87)...(752) 1cgcgggagct gcttggaggc
tcggcggccg ggaggaggcc ggggccacgc ttcttggaag 60ctactgagtg acttctttga
agaacc atg aag tca cac tat att gtg cta gct 113 Met Lys Ser His Tyr
Ile Val Leu Ala 1 5cta gcc tcc ctg acg ttc ctg ctg tgt ctc ccc gtg
tcc cag agc tgt 161Leu Ala Ser Leu Thr Phe Leu Leu Cys Leu Pro Val
Ser Gln Ser Cys 10 15 20 25aac aaa gca ctc tgt gcc agc gat gtg agc
aaa tgc ctc att cag gag 209Asn Lys Ala Leu Cys Ala Ser Asp Val Ser
Lys Cys Leu Ile Gln Glu 30 35 40ctc tgc cag tgc cgg cct gga gaa ggg
aac tgc ccc tgc tgt aag gag 257Leu Cys Gln Cys Arg Pro Gly Glu Gly
Asn Cys Pro Cys Cys Lys Glu 45 50 55tgc atg ctg tgc ctc ggg gcc ctg
tgg gac gag tgc tgc gac tgt gtc 305Cys Met Leu Cys Leu Gly Ala Leu
Trp Asp Glu Cys Cys Asp Cys Val 60 65 70ggt atg tgc aac cct cgg aat
tac agc gac acc ccg ccc aca tcc aag 353Gly Met Cys Asn Pro Arg Asn
Tyr Ser Asp Thr Pro Pro Thr Ser Lys 75 80 85agc acc gtg gag gag ctg
cac gag ccc att ccg tcc ctg ttc agg gcg 401Ser Thr Val Glu Glu Leu
His Glu Pro Ile Pro Ser Leu Phe Arg Ala 90 95 100 105ctg acg gag
ggc gac acc cag ctg aac tgg aac atc gtc tcc ttc cct 449Leu Thr Glu
Gly Asp Thr Gln Leu Asn Trp Asn Ile Val Ser Phe Pro 110 115 120gtg
gca gag gag ctg tca cac cat gaa aac cta gtc tcc ttc cta gaa 497Val
Ala Glu Glu Leu Ser His His Glu Asn Leu Val Ser Phe Leu Glu 125 130
135act gtg aac cag ctg cac cac caa aac gtg tct gtt ccc agc aac aat
545Thr Val Asn Gln Leu His His Gln Asn Val Ser Val Pro Ser Asn Asn
140 145 150gtc cac gcc ccc ttc ccc agc gac aaa gag cgc atg tgc aca
gtg gtt 593Val His Ala Pro Phe Pro Ser Asp Lys Glu Arg Met Cys Thr
Val Val 155 160 165tac ttt gat gac tgc atg tcc atc cac cag tgt aag
ata tcc tgc gaa 641Tyr Phe Asp Asp Cys Met Ser Ile His Gln Cys Lys
Ile Ser Cys Glu170 175 180 185tcc atg ggt gca tcc aag tat cgc tgg
ttt cac aac gcc tgc tgc gag 689Ser Met Gly Ala Ser Lys Tyr Arg Trp
Phe His Asn Ala Cys Cys Glu 190 195 200tgc atc ggt cca gag tgc att
gac tat ggg agt aaa act gtc aag tgt 737Cys Ile Gly Pro Glu Cys Ile
Asp Tyr Gly Ser Lys Thr Val Lys Cys 205 210 215atg aac tgc atg ttt
taaagagggg gaagaaatgc aaaccaaagc agtaagtcat 792Met Asn Cys Met Phe
220gaagtgtgca gaaatcttgg ttctggtatg ctaggagtgt gttaagttat
atgattgtaa 852ctgtgctttt tatatctggt gcctattagt gtaggtcttt
tccattggat tcaatggaac 912tttagtcaca tgaggatcgg gagttcagag
gagtcctggg aaaacctgac atgctgacag 972aaggtgccgt cttcttccag
ctttccaaac acttctcgtt ttgaacgtga tagcacaagc 1032ctggtacatg
tgtggttctc acctgccagt tgtagaacac taggtcccta tagtcacaca
1092tctcttaatt gtgccttggc tggcttacct gttttgtatg agtaaatatt
acagtttata 1152attctaacaa ctcacattca agccatgctg aaacttaatt
tcaaaccact ttacattggt 1212tttagaaagt aaatatttac tatattttac
aacagaagag ttttgcctag ggccagcgag 1272ctgactcagt ggataaaggc
gcttgctacc aagcctgata acctgagttc catccccaga 1332gcccgtacag
tggaaggaca ggaccagctg ctgggagttg tcctctgacc tccagacagg
1392cacagtatca tgcgtggagg tgtgcttgtg tgtgcacaca cataactaac
tgtttttaaa 1452aatataaacc tcttacatgg tgaaatctaa atctgtcgtg
tagctctcac actgacagtg 1512gtttggatgt tatgtcccct gtccgcctgt
agtgctggtg tggtgagaca cagagtcgtc 1572actgctctgg tatagaagag
ttttgtctac caagagtgtc atggcatacc tttggaactt 1632catcaaatgc
acttgaggat gacctgggtc aggaagtagc caggtaaaag cagcgggact
1692gtaggcgatg ctccattaga ctccgtgcag agcagcaggt gcacagcata
gctgggtgtg 1752cggctgacca ggagagggtc tgactccgca ccagcagaac
agcagggtct ccagcacgtg 1812tgggaagcac gtgggagagg gttgaggaag
gatgcacaga tgtggacaga gaagcataaa 1872aatgtcggga actcctagta
gggtccacct taaaatcgct ttatagtctc tggctttgtt 1932actctgtaag
attacacttg tttctggata tctgaatcca aataagcatc atattttaag
1992aagctctgtt tctgaacttc cagggggaaa tctgtttaat gtgtttactc
ctagcatact 2052acagaatttt ctagctctat agcttcttac ctagcgtttc
catagtgctg agcttcatta 2112ctacacgccc ttcctagtaa taaaattctc
accttcaagc atgaatcaaa aacaaatatc 2172tataatacac aggttcaatt
ttatagaatt gctattttct ctagtgcata tctcattaaa 2232agtaactttt
taggaataat ctttatatgg gtacatattt tggtacataa aatagaaaat
2292gttcttaaac tcattttgta ttatttgaat agttacaaga tgatttgtgg
tatcatgggt 2352acccattata aaccatgctc ttcccagtag ctgacgaact
caaggtatca cagccttcta 2412agaagccgac ttagaacatg gctgtacatg
aatattatac attaaggtgt cctctcactt 2472ctacccagag tgcctctgtt
caaaggtgcc ttggaaacat ttcagcccct tccttcttag 2532ctcccacagg
gctgtgggtg ttcttgaaat caggaggcgt tttgaaggac cacagctgct
2592ccatttcagc cgctgattct taggaaagtt catgctctga cagaagtgtg
ctttgatggc 2652ttctagcggt gcatctcgtc tcgttttctt tgtttgtttt
tgttgttgct atcatggttt 2712ggtttggttt tgagacagga tctctgtgca
gccctggctg gcctggaatg tactatgtag 2772accaggctgg ctctcctcat
gttttcttag tgatggccat aaacattgtt aaaatacatc 2832accatctttt
aaaaactttt cattattaaa atttaaaata tagcatgtca tttttttacc
2892ccatacattt gctatgaaaa attttttaaa ccacctgctt taactttttt
attgccctgt 2952ttttcctatt agaattgatc cccactgagg taaattttat
aatcatgttt tgtgtatttt 3012tcctggctcg ccaaggctta tgaagaaata
gcagccattc cctgacaggt ttgcgctccc 3072accacagaga ggctgagcaa
gatgatcaga ggatcaaggc cagccagagc aaggcactgc 3132ccagaaagca
caagtcctgt gctcagcgtt ttgcgtagcg ttttattcct aattgaaatg
3192taatatttca gaagctagca gcctcgctca gtctagacct tccacaccaa
tctagcagcg 3252attctcccgt actaaagcct ttgtaagagt ttacggttct
tcctcagtga aaaatgatct 3312tgtttttctt acagccggat ccaaagacgc
tagatgttaa gggctgaggc tgaagcccgg 3372tgacggggcg ctcacctgtc
atggtgcagc cctcgttcca ccgtgagcac cagcaagaga 3432caaacacaag
cttgtgagtc agaggccgtt attaaattca tacgcacata ctccctatag
3492cgagacatgg gcttatgggc aggctttttt tttcataaca tttatgagaa
aacaatgttt 3552tccccataac atttaattag gactgtagct tattggtaat
taaggtacaa aatcaaagtc 3612gagtagaatg tactgttcac acagcgtgtt
gtgaaagggg tcctcacacc aaagtttaac 3672tgtaaagttt agaaaaataa
cattgtcatt agcatatttg aacacatatt tggaatttct 3732aaaaagcatc
aaaatagaaa aagaaagtga aactctggag aatgagatgc tgaagatggg
3792ctatgattta aaggtctgtt ctgtagttag aaagcacctt ttaaagactt
tgttcattcc 3852caagagtcta tgttgattgc atttaacatg accgacaact
tatatatgta attgtgtaca 3912ttttcattgg ttgtctctgt agtccaaaag
aaggtatttt aataaaaaat agaaatgact 3972gtgaaaaaaa aaaa 39862222PRTMus
musculus 2Met Lys Ser His Tyr Ile Val Leu Ala Leu Ala Ser Leu Thr
Phe Leu 1 5 10 15Leu Cys Leu Pro Val Ser Gln Ser Cys Asn Lys Ala
Leu Cys Ala Ser 20 25 30Asp Val Ser Lys Cys Leu Ile Gln Glu Leu Cys
Gln Cys Arg Pro Gly 35 40 45Glu Gly Asn Cys Pro Cys Cys Lys Glu Cys
Met Leu Cys Leu Gly Ala 50 55 60Leu Trp Asp Glu Cys Cys Asp Cys Val
Gly Met Cys Asn Pro Arg Asn65 70 75 80Tyr Ser Asp Thr Pro Pro Thr
Ser Lys Ser Thr Val Glu Glu Leu His 85 90 95Glu Pro Ile Pro Ser Leu
Phe Arg Ala Leu Thr Glu Gly Asp Thr Gln 100 105 110Leu Asn Trp Asn
Ile Val Ser Phe Pro Val Ala Glu Glu Leu Ser His 115 120 125His Glu
Asn Leu Val Ser Phe Leu Glu Thr Val Asn Gln Leu His His 130 135
140Gln Asn Val Ser Val Pro Ser Asn Asn Val His Ala Pro Phe Pro
Ser145 150 155 160Asp Lys Glu Arg Met Cys Thr Val Val Tyr Phe Asp
Asp Cys Met Ser 165 170 175Ile His Gln Cys Lys Ile Ser Cys Glu Ser
Met Gly Ala Ser Lys Tyr 180 185 190Arg Trp Phe His Asn Ala Cys Cys
Glu Cys Ile Gly Pro Glu Cys Ile 195 200 205Asp Tyr Gly Ser Lys Thr
Val Lys Cys Met Asn Cys Met Phe 210 215 220319DNAArtificial
SequenceArtificially Synthesized Primer Sequence 3gggggtggac
catcctcta 19420DNAArtificial SequenceArtificially Synthesized
Primer Sequence 4cgcgcagctg taaacggtag 205225PRTDrosophila
melanogaster 5Met Gln Leu Leu Cys Tyr Phe Val Ile Leu Phe Val Gly
Ile Ala Pro 1 5 10 15Trp Ser Ser Leu Ala Asn Asp Asp Gly Cys Asn
Glu Val Val Cys Gly 20 25 30Ser Val Val Ser Lys Cys Leu Ile Thr Gln
Ser Cys Gln Cys Lys Leu 35 40 45Asn Asp Cys His Cys Cys Lys Asp Cys
Leu Asn Cys Leu Gly Glu Leu 50 55 60Tyr Ile Glu Cys Cys Gly Cys Leu
Asp Met Cys Pro Lys His Lys Asp65 70 75 80Val Leu Pro Ser Leu Thr
Pro Arg Ser Glu Ile Gly Asp Ile Glu Gly 85 90 95Val Pro Glu Leu Phe
Asp Thr Leu Thr Ala Glu Asp Asp Glu Gly Trp 100 105 110Ser Thr Ile
Arg Phe Ser Met Arg Ala Gly Phe Lys Gln Arg Val Gln 115 120 125Gly
Gly Ala Ser Gly Asp Ala Gly Asn Gly Asn Gly Asn Gly Asn Ala 130 135
140Gly Ser Ala Gly Val Thr Leu Cys Thr Val Ile Tyr Val Asn Ser
Cys145 150 155 160Ile Arg Ala Asn Lys Cys Arg Gln Gln Cys Glu Ser
Met Gly Ala Ser 165 170 175Ser Tyr Arg Trp Phe His Asp Gly Cys Cys
Glu Cys Val Gly Glu Asn 180 185 190Cys Leu Asn Tyr Gly Ile Asn Glu
Ser Arg Cys Arg Gly Cys Pro Glu 195 200 205Asp Gln Asp Gln Leu Leu
Thr Ala Asp Thr Val Pro Ala Glu Ala Glu 210 215 220Gln225
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