U.S. patent application number 11/048402 was filed with the patent office on 2005-10-20 for human cdnas encoding polypeptides having kinase functions.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Anderson, Dirk M., Bird, Timothy A., Marken, John S., Virca, G. Duke.
Application Number | 20050233355 11/048402 |
Document ID | / |
Family ID | 26790022 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233355 |
Kind Code |
A1 |
Virca, G. Duke ; et
al. |
October 20, 2005 |
Human cDNAs encoding polypeptides having kinase functions
Abstract
The invention is directed to purified and isolated human
polypeptides having kinase function, the nucleic acids encoding
such polypeptides, processes for production of recombinant forms of
such polypeptides, antibodies generated against these polypeptides,
fragmented peptides derived from these polypeptides, the use of
such polypeptides and fragmented peptides in phosphorylation
reactions and as molecular weight markers, the use of such
polypeptides and fragmented peptides as controls for peptide
fragmentation, the use of such polypeptides in screening assays,
and kits comprising these reagents.
Inventors: |
Virca, G. Duke; (Seattle,
WA) ; Bird, Timothy A.; (Seattle, WA) ;
Anderson, Dirk M.; (Seattle, WA) ; Marken, John
S.; (Seattle, WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
LAW DEPARTMENT
1201 AMGEN COURT WEST
SEATTLE
WA
98119
US
|
Assignee: |
Immunex Corporation
|
Family ID: |
26790022 |
Appl. No.: |
11/048402 |
Filed: |
January 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11048402 |
Jan 31, 2005 |
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10024828 |
Dec 18, 2001 |
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6867013 |
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10024828 |
Dec 18, 2001 |
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09509902 |
Jun 23, 2000 |
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6387676 |
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09509902 |
Jun 23, 2000 |
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PCT/US99/17630 |
Aug 3, 1999 |
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60095270 |
Aug 4, 1998 |
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60099972 |
Sep 11, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1205
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/12; C12N 015/09 |
Claims
What is claimed is:
1. An isolated polynucleotide molecule selected from: a) a
nucleotide sequence encoding a polypeptide having kinase activity
comprising a sequence having at least 80% identity to the
polypeptide sequence of SEQ ID NO:9; b) a nucleotide sequence
encoding a polypeptide having kinase activity comprising a sequence
having at least 90% identity to the polypeptide of SEQ ID NO:9; c)
degenerate nucleotide sequences encoding a polypeptide of a) or b);
d) the nucleotide sequence of SEQ ID NO:3; e) nucleotide sequences
capable of hybridization to nucleotide of a) or b) under conditions
of moderate stringency; f) nucleotide sequences complementary to
a), b), c), d) or e).
2. An isolated polynucleotide molecule encoding a polypeptide
comprising the sequence of SEQ ID NO:9.
3. A recombinant vector that directs the expression of the
polynucleotide molecule of claim 2.
4. An isolated polypeptide having kinase activity encoded by the
polynucleotide molecule of claim 1.
5. An isolated polypeptide according to claim 4 having a molecular
weight of approximately 39,284 Daltons as determined by
SDS-PAGE.
6. An isolated polypeptide according to claim 4 in non-glycosylated
form.
7. A recombinant host cell comprising the polynucleotide of claim
2.
8. A method for the production of a polypeptide encoded by the
polynucleotide of claim 2 comprising culturing a recombinant host
cell comprising the polynucleotide of claim 2 under conditions
promoting expression of said polypeptide.
9. The method of claim 8, wherein the host cell is selected from
the group consisting of bacterial cells, yeast cells, plant cells,
insect cells, and animal cells.
10. An isolated polypeptide having kinase activity produced by the
method of claim 8.
11. An isolated polypeptide having kinase activity selected from:
a) a polypeptide comprising a sequence having at least 80% identity
to the polypeptide sequence of SEQ ID NO:9; b) a polypeptide
comprising a sequence having at least 80% identity to the
polypeptide sequence of SEQ ID NO:9; c) a polypeptide sequence
comprising SEQ ID NO:9
12. Isolated antibodies that bind to a polypeptide of claim 11.
13. A method of screening a candidate molecule to identify its
ability to inhibit (antagonize) or agonize a polypeptide encoded by
a polynucleotide of claim 2, said method comprising the steps of:
a) adding said candidate molecule to a medium which contains cells
expression said polypeptide; b) determining the level of biological
activity of said polypeptide in said medium; and c) comparing the
level of biological activity of step (b) with the level of
biological activity that occurs in said medium in the absence of
said candidate molecule; wherein a decreased level of biological
activity of step (b) as compared to the level of biological
activity that occurs in said medium in the absence of said
candidate molecule indicates an antagonist and an increased level
of biological activity indicates an agonist.
14. A method of screening a candidate molecule to identify its
ability to mimic the biological activity of a polypeptide encoded
by a polynucleotide of claim 2, said method comprising the steps
of: a) adding said candidate molecule to a biological assay to
determine its biological effects; and b) comparing said biological
effects to said candidate molecule with the biological effects of
said polypeptide.
15. A method for screening a candidate molecule for its ability to
bind to a polypeptide, the method comprising determining if said
candidate molecule binds to a polypeptide of claim 11.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
10/024,828, filed Dec. 18, 2001; which is a divisional of U.S. Ser.
No. 09/509,902, having a filing date under 35 U.S.C. .sctn. 102(e)
of Jun. 23, 2000 and issued as U.S. Pat. No. 6,387,676, on May 14,
2002; which is a national application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US99/17630, having an
international filing date of 3 Aug. 1999 and published in English
on Feb. 17, 2000; which claims the priority of provisional
applications U.S. Ser. No. 60/095,270, filed Aug. 4, 1998, and U.S.
Ser. No. 60/099,972, filed Sep. 11, 1998; all of which are
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention is directed to purified and isolated human
polypeptides having kinase function, the nucleic acids encoding
such polypeptides, processes for production of recombinant forms of
such polypeptides, antibodies generated against these polypeptides,
fragmented peptides derived from these polypeptides, the use of
such polypeptides and fragmented peptides in phosphorylation
reactions and as molecular weight markers, the use of such
polypeptides and fragmented peptides as controls for peptide
fragmentation, the use of such polypeptides in screening assays,
and kits comprising these reagents.
BACKGROUND OF THE INVENTION
[0003] The eukaryotic protein kinases make up a large and rapidly
expanding family of proteins related on the basis of homologous
catalytic domains. Spurred by the development of gene cloning and
sequencing methodologies, distinct protein kinase genes have been
identified from a wide selection of invertebrates and lower
eukaryotes, including Drosophila, Caenorhabditis elegans, Aplysia,
Hydra, Dictyostelium, and budding (Saccharomyces cerevisiae) and
fission (Schizosaccharomyces pombe) yeast. Homologous genes have
also been identified in higher plants. Protein kinases, however,
are not limited to the eukaryotes. Enzyme activities have been well
documented in prokaryotes, but the prokaryotic protein kinase genes
are not obviously homologous to those of the eukaryotes. Because
protein kinases are useful biochemical reagents, there is a need in
the art for the continued discovery of unique members of the
protein kinase family.
[0004] In addition, the discovery and identification of proteins
are at the forefront of modern molecular biology and biochemistry.
The identification of the primary structure, or sequence, of a
sample protein is the culmination of an arduous process of
experimentation. In order to identify an unknown sample protein,
the investigator can rely upon comparison of the unknown sample
protein to known peptides using a variety of techniques known to
those skilled in the art. For instance, proteins are routinely
analyzed using techniques such as electrophoresis, sedimentation,
chromatography, and mass spectrometry.
[0005] Comparison of an unknown protein sample to polypeptides of
known molecular weight allows a determination of the apparent
molecular weight of the unknown protein sample (T. D. Brock and M.
T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed.
1991)). Protein molecular weight standards are commercially
available to assist in the estimation of molecular weights of
unknown protein samples (New England Biolabs Inc. Catalog:130-131,
1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the
molecular weight standards may not correspond closely enough in
size to the unknown sample protein to allow an accurate estimation
of apparent molecular weight.
[0006] The difficulty in estimation of molecular weight is
compounded in the case of proteins that are subjected to
fragmentation by chemical or enzymatic means (A. L. Lehninger,
Biochemistry 106-108 (Worth Books, 2d ed. 1981)). Chemical
fragmentation can be achieved by incubation of a protein with a
chemical, such as cyanogen bromide, which leads to cleavage of the
peptide bond on the carboxyl side of methionine residues (E. Gross,
Methods in Enz. 11:238-255, 1967). Enzymatic fragmentation of a
protein can be achieved by incubation of a protein with a protease
that cleaves at multiple amino acid residues (D. W. Cleveland et
al., J. Biol. Chem. 252:1102-1106, 1977). Enzymatic fragmentation
of a protein can also be achieved by incubation of a protein with a
protease, such as Achromobacter protease I (F. Sakiyama and A.
Nakata, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.
Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys.
Acta 660:51-55, 1981), which leads to cleavage of the peptide bond
on the carboxyl side of lysine residues. The molecular weights of
the fragmented peptides can cover a large range of molecular
weights and the peptides can be numerous. Variations in the degree
of fragmentation can also be accomplished (D. W. Cleveland et al.,
J. Biol. Chem. 252:1102-1106, 1977).
[0007] The unique nature of the composition of a protein with
regard to its specific amino acid constituents results in a unique
positioning of cleavage sites within the protein. Specific
fragmentation of a protein by chemical or enzymatic cleavage
results in a unique "peptide fingerprint" (D. W. Cleveland et al.,
J. Biol. Chem. 252:1102-1106, 1977; M. Brown et al., J. Gen. Virol.
50:309-316, 1980). Consequently, cleavage at specific sites results
in reproducible fragmentation of a given protein into peptides of
precise molecular weights. Furthermore, these peptides possess
unique charge characteristics that determine the isoelectric pH of
the peptide. These unique characteristics can be exploited using a
variety of electrophoretic and other techniques (T. D. Brock and M.
T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed.
1991)).
[0008] When a peptide fingerprint of an unknown protein is
obtained, this can be compared to a database of known proteins to
assist in the identification of the unknown protein (W. J. Henzel
et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; B. Thiede et
al., Electrophoresis 1996, 17:588-599, 1996). A variety of computer
software programs are accessible via the Internet to the skilled
artisan for the facilitation of such comparisons, such as
MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html),
PeptideSearch (Internet site: www.mann.emblheiedelberg.de . . .
deSearch/FR_PeptideSearchForm.html), and ProFound (Internet site:
www.chait-sgi.rockefeller.edu/cgi-bin/prot-i- d-frag.html). These
programs allow the user to specify the cleavage agent and the
molecular weights of the fragmented peptides within a designated
tolerance. The programs compare these molecular weights to protein
databases to assist in the elucidation of the identity of the
sample protein. Accurate information concerning the number of
fragmented peptides and the precise molecular weight of those
peptides is required for accurate identification. Therefore,
increasing the accuracy in the determination of the number of
fragmented peptides and the precise molecular weight of those
peptides should result in enhanced success in the identification of
unknown proteins.
[0009] Fragmentation of proteins is further employed for the
production of fragments for amino acid composition analysis and
protein sequencing (P. Matsudiara, J. Biol. Chem. 262:10035-10038,
1987; C. Eckerskorn et al., Electrophoresis 1988, 9:830-838, 1988),
particularly the production of fragments from proteins with a
"blocked" N-terminus. In addition, fragmentation of proteins can be
used in the preparation of peptides for mass spectrometry (W. J.
Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; B.
Thiede et al., Electrophoresis 1996, 17:588-599, 1996), for
immunization, for affinity selection (R. A. Brown, U.S. Pat. No.
5,151,412), for determination of modification sites (e.g.
phosphorylation), for generation of active biological compounds (T.
D. Brock and M. T. Madigan, Biology of Microorganisms 300-301
(Prentice Hall, 6d ed. 1991)), and for differentiation of
homologous proteins (M. Brown et al., J. Gen. Virol. 50:309-316,
1980).
[0010] In view of the continuing interest in protein research and
the elucidation of protein structure and properties, there exists a
need in the art for polypeptides having kinase function or suitable
for use in peptide fragmentation studies and in molecular weight
measurements.
SUMMARY OF THE INVENTION
[0011] The invention aids in fulfilling these needs in the art. The
invention encompasses an isolated human nucleic acid molecule
comprising the DNA sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 13, or
15 and an isolated human nucleic acid molecule encoding the amino
acid sequence of SEQ ID NO:7, 8,9, 10, 11, 12, 14, or 16. The
invention also encompasses nucleic acid molecules complementary to
these sequences. As such, the invention includes double-stranded
nucleic acid molecules comprising the DNA sequence of SEQ ID NO:1,
2, 3, 4, 5, 6, 13, or 15 and isolated nucleic acid molecules
encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12,
14 or 16. Both single-stranded and double-stranded RNA and DNA
nucleic acid molecules are encompassed by the invention. These
molecules can be used to detect both single-stranded and
double-stranded RNA and DNA variants encompassed by the invention.
A double-stranded DNA probe allows the detection of nucleic acid
molecules equivalent to either strand of the nucleic acid molecule.
Isolated nucleic acid molecules that hybridize to a denatured,
double-stranded DNA comprising the DNA sequence of SEQ ID NO:1, 2,
3, 4, 5, 6, 13, or 15, or an isolated nucleic acid molecule
encoding the amino acid sequence of SEQ ID NO:7, 8, 9, 10, 11, 12,
14, or 16 are within the invention. A preferred set of
hybridization conditions are those of moderate stringency: in 50%
formamide and 6.times.SSC, at 42.degree. C. with washing conditions
of 60.degree. C., 0.5.times.SSC, 0.1% SDS.
[0012] The invention further encompasses isolated nucleic acid
molecules derived by in vitro mutagenesis from SEQ ID NO:1, 2, 3,
4, 5, 6, 13, or 15. In vitro mutagenesis would include numerous
techniques known in the art including, but not limited to,
site-directed mutagenesis, random mutagenesis, and in vitro nucleic
acid synthesis. The invention also encompasses isolated nucleic
acid molecules degenerate from SEQ ID NO:1, 2, 3, 4, 5, or 6 (and
the resulting amino acid sequence) as a result of the genetic code,
isolated nucleic acid molecules that are allelic variants of human
DNA of the invention, or a species homolog of DNA of the invention.
The invention also encompasses recombinant vectors that direct the
expression of these nucleic acid molecules and host cells
transformed or transfected with these vectors. In addition, the
invention encompasses methods of using the nucleic acid noted above
in assays to identify chromosomes, map human genes, and study
tumors.
[0013] The invention also encompasses isolated polypeptides encoded
by these nucleic acid molecules, including isolated polypeptides
having a molecular weights as determined by SDS-PAGE, isolated
polypeptides in non-glycosylated form, and fragments thereof. The
invention further includes synthetic polypeptides encoded by these
nucleic acid molecules. Peptides and fragments of these
polypeptides, however derived, are also part of the invention and
may be produced by any standard means, from chemical, enzymatic,
recombinant, or synthetic methods. Isolated polyclonal or
monoclonal antibodies that bind to these polypeptides are
encompassed by the invention. The invention further encompasses
methods for the production of polypeptides having kinase functions
including culturing a host cell under conditions promoting
expression and recovering the polypeptide from the culture medium.
Especially, the expression of polypeptides having kinase functions
in bacteria, yeast, plant, insect, and animal cells is encompassed
by the invention.
[0014] In general, the polypeptides of the invention having kinase
function can be used to phosphorylate target proteins and to
radiolabeled target proteins with .sup.32P. In addition, the
polypeptides of the invention having kinase function can be used to
identify proteins having a phosphate activity.
[0015] In addition, assays utilizing polypeptides having kinase
functions to screen for potential inhibitors of activity associated
with polypeptide counter-structure molecules, and methods of using
polypeptides having kinase functions as therapeutic agents for the
treatment of diseases mediated by polypeptide counter-structure
molecules are encompassed by the invention. Methods of using
polypeptides having kinase functions in the design of inhibitors
thereof are also an aspect of the invention. The invention further
encompasses use of polypeptides of the invention to screen for
agonists and antagonists.
[0016] The invention further encompasses the fragmented peptides
produced from polypeptides of the invention by chemical or
enzymatic treatment. In addition, the polypeptides of the invention
and fragmented peptides thereof, wherein at least one of the sites
necessary for fragmentation by chemical or enzymatic means has been
mutated, are an aspect of the invention.
[0017] The invention further includes a method for using these
polypeptides and fragmented peptides thereof as molecular weight
markers that allow the estimation of the molecular weight of a
protein or a fragmented protein sample. The invention also
encompasses a method for the visualization of the molecular weight
markers of the invention thereof using electrophoresis. The
invention further encompasses methods for using the polypeptides of
the invention and fragmented peptides thereof as markers, which aid
in the determination of the isoelectric point of a sample protein.
The invention also encompasses methods for using polypeptides of
the invention and fragmented peptides thereof as controls for
establishing the extent of fragmentation of a protein sample.
[0018] Further encompassed by this invention are kits to aid the
determination of molecular weights of a sample protein utilizing
polypeptide molecular weight markers of the invention, fragmented
peptides thereof, and forms of these polypeptide molecular weight
markers, wherein at least one of the sites necessary for
fragmentation by chemical or enzymatic means has been mutated.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The protein kinases are a large family of enzymes, many of
which mediate the response of eukaryotic cells to external stimuli.
In recent years, members of the protein kinase family have been
discovered at an accelerated pace. The surge in the number of known
protein kinases has been largely due to the advent of gene cloning
and sequencing techniques. Amino acid sequences deduced from
nucleotide sequences are considered to represent protein kinases if
they include certain key residues that are highly conserved in the
protein kinase catalytic domain. A cDNA encoding a human
polypeptide has been isolated and is set forth in SEQ ID NO:1, 2,
3, 4, 5, 6, 13, or 15. This discovery of the cDNA encoding human
polypeptides having kinase functions enables construction of
expression vectors comprising nucleic acid sequences encoding
polypeptides having kinase functions; host cells transfected or
transformed with the expression vectors; biologically active human
polypeptides having kinase functions, and molecular weight markers
as isolated and purified proteins; and antibodies immunoreactive
with polypeptides of the invention.
[0020] More particularly, the invention relates to certain
nucleotide sequences. A "nucleotide sequence" refers to a
polynucleotide molecule in the form of a separate fragment or as a
component of a larger nucleic acid construct, that has been derived
from DNA or RNA isolated at least once in substantially pure form
(i.e., free of contaminating endogenous materials) and in a
quantity or concentration enabling identification, manipulation,
and recovery of its component nucleotide sequences by standard
biochemical methods (such as those outlined in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences are
preferably provided and/or constructed in the form of an open
reading frame uninterrupted by internal non-translated sequences,
or introns, that are typically present in eukaryotic genes.
Sequences of non-translated DNA can be present 5' or 3' from an
open reading frame, where the same do not interfere with
manipulation or expression of the coding region.
[0021] Particularly preferred nucleotide sequences of the invention
include the following:
1 NAME: HH0900-BF04 DNA Nucleotide sequence: (SEQ ID NO:1)
GTACGCCATGAAGGTGCTGCGCAAGGCGGCGCTGGTGCAGCGCGCCAAGA
CGCAAGAGCACACGCGCACCGAGCGCTCGGTGCTGGAGCTGGTGCGCCAG
GCGCCCTTCCTGGTCACGCTGCACTACGCTTTCCAGACGGATGCCAAGCT
GCACCTCATCCTGGACTATGTGAGCGGCGGG; NAME: HH2040-BF04 DNA Nucleotide
sequence: (SEQ ID NO:2)
CCCGAGAGGTGCCACATCAGACCGCCTCCGACTTCGTGCGGGACTCGGCG
GCCAGCCACCAGGCGGAGCCCGAGGCGTACGAGCGGCGCGTGTGCTTCCT
GCTTCTGCAACTCTGCAACGGGCTGGAGCACCTGAAGGAGCACGGGATCA
TCCACCGGGACCTGTGCCTGGAGAACCTGCTGCTGGTGCACTGCACCCTC
CAGGCCGGCCCCGGGCCCGCC; Name: JJ503-KS DNA nucleotide sequence (SEQ
ID NO:3) CGGGCAGGGCTGGAGCTGGGCTGG-
GATCCCGAGCTCGGCAGCAGCGCAGCGGGCCGGCCCACCTGCTGGTGC
CCTGGAGGCTCTGAGCCCCGGCGGCGCCCGGGCCCACGCGGAACGACGGGGCGAGATGCGAGCCACCCCTCT
GGCTGCTCCTGCGGGTTCCCTGTCCAGGAAGAAGCGGTTGGAGTTGGATGACAACTTAGATACCGA-
GCGTCC
CGTCCAGAAACGAGCTCGAAGTGGGCCCCAGCCCAGACTGCCCCCCTGCCTGTTGCCCCT-
GAGCCCACCTAC
TGCTCCAGATCGTGCAACTGCTGTGGCCACTGCCTCCCGTCTTGGGCCCTATGT-
CCTCCTGGAGCCCGAGGA
GGGCGGGCGGGCCTACCAGGCCCTGCACTGCCCTACACGCACTGAGTA-
TACCTGCAAGGTGTACCCCGTCCA
GGAAGCCCTGGCCGTGCTGGAGCCCTACGCGCGGCTGCCCCC-
GCACAAGCATGTGGCTCGGCCCACTGAGGT
CCTGGCTGGTACCCAGCTCCTCTACGCCTTTTTCAC-
TCGGACCCATGGGGACATGCACAGCCTGGTGCGAAG
CCGCCACCGTATCCCTGAGCCTGAGGCTGC-
CGTGCTCTTCCGCCAGATGGCCACCGCCCTGGCGCACTGTCA
CCAGCACGGTCTGGTCCTGCGTGA-
TCTCAAGCTGTGTCGCTTTGTCTTCGCTGACCGTGAGAGGAAGAAGCT
GGTGCTGGAGAACCTGGAGGACTCCTGCGTGCTGACTGGGCCAGATGATTCCCTGTGGGACAAGCACGCGTG
CCCAGCCTACGTGGGACCTGAGATACTCAGCTCACGGGCCTCATACTCGGGCAAGGCAGCCGATGT-
CTGGAG
CCTGGGCGTGGCGCTCTTCACCATGCTGGCCGGCCACTACCCCTTCCAGGACTCGGAGCC-
TGTCCTGCTCTT
CGGCAAGATCCGCCGCGGGGCCTACGCCTTGCCTGCAGGCCTCTCGGCCCCTGC-
CCGCTGTCTGGTTCGCTG
CCTCCTTCGTCGGGAGCCAGCTGAACGGCTCACAGCCACAGGCATCCT-
CCTGCACCCCTGGCTGCGACAGGA CCCGA; Name: QQ1249-BF04 DNA Nucleotide
sequence: (SEQ ID NO:4)
CAGCGAGAAGCCGACATGCATCGCCTCTTCAATCACCCCAACATCCTTCG
CCTCGTGGCTTACTGTCTGAGGGAACGGGGTGCTAAGCATGAGGCCTGGC
TGCTGCTACCATTCTTCAAGAGAGGTACGCTGTGGAATGAGATAGAAAGG
CTGAAGGACAAAGGCAACTTCCTGACCGAGGATCAAATCCTTTGGCTGCT
GCTGGGGATCTGCAGAGGCCTTGAGGCCATTCATGCCAAGGGTTATGCCT
ACAGAGACTTGAAGCCCACCAATATATTGCTTGGAGATGAGGGGCAGCCA
GTTTTAATGGACTTGGGTTCCATGAATCAAGCATGCATCCATGTGGAGGG
CTCCCGCCAGGCTCTGACCCTGCAGGACTGGGCAGCCC; Name: QQ3351-BF04 DNA
Nucleotide sequence: (SEQ ID NO:5)
ATGCTAACTAGTTTAAACAGATCTTGGAACGAGACGACCTGCTGTGGAAGAGCGAGCTTTTTGGAACTGTGC
ACGGGACAGATTGGACGCACACCCCTCGGGAGGCGCGAAGGCATGGAAAATTTGAAGCATATTATC-
ACCCTT
GGCCAGGTCATCCACAAACGGTGTGAAGAGATGAAATACTGCAAGAAACAGTGCCGGCGC-
CTGGGCCACCGC
GTCCTCGGCCTGATCAAGCCTCTGGAGATGCTCCAGGACCAAGGAAAGAGGAGC-
GTGCCCTCTGAGAAGTTA
ACCACAGCCATGAACCGCTTCAAGGCTGCCCTGGAGGAGGCTAATGGG-
GAGATAGAAAAGTTCAGCAATAGA
TCCAATATCTGCAGGTTTCTAACAGCAAGCCAGGACAAAATA-
CTCTTCAAGGACGTGAACAGGAAGCTGAGT
GATGTCTGGAAGGAGCTCTCGCTGTTACTTCAGGTT-
GAGCAACGCATGCCTGTTTCACCCATAAGCCAAGGA
GCGTCCTGGGCACAGGAAGATCAGCAGGAT-
GCAGACGAAGACAGGCGAGCTTTCCAGATGCTAAGAAGAGAT
AATGAAAAAATAGAAGCTTCACTG-
AGACGATTAGAAATCAACATGAAAGAAATCAAGGAAACTTTGAGGCAG
TATTTACCACCAAAATGCATGCAGGAGATCCCGCAAGAGCAAATCAAGGAGATCAAGAAGGAGCAGCTTTCA
GGATCCCCGTGGATTCTGCTAAGGGAAAATGAAGTCAGCACACTTTATAAAGGAGAATACCACAGA-
GCTCCA
GTGGCCATAAAAGTATTCAAAAAACTCCAGGCTGGCAGCATTGCAATAGTGAGGCAGACT-
TTCAATAAGGAG
ATCAAAACCATGAAGAAATTCGAATCTCCCAACATCCTGCGTATATTTGGGATT-
TGCATTGATGAAACAGTG
ACTCCGCCTCAATTCTCCATTGTCATGGAGTACTGTGAACTCGGGACC-
CTCAGGGAGCTGTTGGATAGGGAA
AAAGACCTCACACTTGGCAAGCGCATGGTCCTAGTCCTGGGG-
GCAGCCCGAGGCCTATACCGGCTACACCAT
TCAGAAGCACCTGAACTCCACGGAAAAATCAGAAGC-
TCAAACTTCCTGGTAACTCAAGGCTACCAAGTGAAG
CTTGCAGGATTTGAGTTGAGGAAAACACAG-
ACTTCCATGAGTTTGGGAACTACGAGAGAAAAGACAGACAGA
GTCAAATCTACAGCATATCTCTCA-
CCTCAGGAACTGGAAGATGTATTTTATCAATATGATGTAAAGTCTGAA
ATATACAGCTTTGGAATCGTCCTCTGGGAAATCGCCACTGGAGATATCCCGTTTCAAGGCTGTAATTCTGAG
AAGATCCGCAAGCTGGTGGCTGTGAAGCGGCAGCAGGAGCCACTGGGTGAAGACTGCCCTTCAGAG-
CTGCGG
GAGATCATTGATGAGTGCCGGGCAGCAGGTCGTCTCGTTCCAAGATCTGTAGCGGCCGCC-
CGGGCCGTCGAC GTTTAAACGCGTGGCCCTCGAGAGGTTTTCCGATCCGGTCGAT, and Name:
SS1771 Nucleotide sequence: (SEQ ID NO:6) CTTCCCGCTG GACGTGGAGT
ACGGAGGCCC AGACCGGAGG TGCCCGCCTC CGCCCTACCC GAAGCACCTG CTGCTGCGCA
GCAAGTCGGA GCAGTACGAC CTGGACAGCC TGTGCGCAGG CATGGAGCAG AGCCTCCGTG
CGGGCCCCAA CGAGCCCGAG GGCGGCGACA AGAGCCGCAA AAGCGCCAAG GGGGACAAAG
GCGGAAAGGA TAAAAAGCAG ATTCAGACCT CTCCCGTTCC CGTCCGCAAA AACAGCAGAG
ACGAAGAGAA GAGAGAGTCA CGCATCAAGA GCTACTCGCC ATACGCCTTT AAGTTCTTCA
TGGAGCAGCA CGTGGAGAAT GTCATCAAAA CCTACCAGCA GAAGGTTAAC CGGAGGCTGC
AGCTGGAGCA AGAAATGGCC AAAGCTGGAC TCTGTGAAGC TGAGCAGGAG CAGATGCGGA
AGATCCTCTA CCAGAAAGAG TCTAATTACA ACAGGTTAAA GAGGGCCAAG ATGGACAAGT
CTATGTTTGT CAAGATCAAA ACCCTGGGGA TCCGTGCCTT TGGAGAAGTG TGCCTTGCTT
GTAAGGTGGA CACTCACGCC CTGTACGCCA TGAAGACCCT AAGGAAAAAG GATGTCCTGA
ACCGGAATCA GGTGGCCCAC GTCAAGGCCG AGAGGGACAT CCTGGCCGAG GCAGACAATG
AGTGGGTGGT CAAACTCTAC TACTCCTTCC AAGACAAAGA CAGCCTGTAC TTTGTGATGG
ACTACATCCC TGGTGGGGAC ATGATGAGCC TGCTGATCCG GATGGAGGTC TTCCCTGAGC
ACCTGGCCCG GTTCTACATC GCAGAGCTGA CTTTGGCCAT TGAGAGTGTC CACAAGATGG
GCTTCATCCA CCGAGACATC AAGCCTGATA ACATTTTGAT AGATCTGGAT GGTCACATTA
AACTCACAGA TTTCGGCCTC TGCACTGGGT TCAGGTGGAC TCACAATTCC AAATATTACC
AGAAAGGGAG CCATGTCAGA CAGGACAGCA TGGAGCCCAG CGACCTCTGG GATGATGTGT
CTAACTGTCG GTGTGGGGAC AGGCTGAAGA CCCTAGAGCA GAGGGCGCGG AAGCAGCACC
AGAGGTGCCT GGCACATTCA CTGGTGGGGA CTCCAAACTA CATCGCACCC GAGGTGCTCC
TCCGCAAAGG GTACACTCAA CTCTGTGACT GGTGGAGTGT TGGAGTGATT CTCTTCGAGA
TGCTGGTGGG GCAGCCGCCC TTTTTGGCAC CTACTCCCAC AGAAACCCAG CTGAAGGTGA
TCAACTGGGA GAACACGCTC CACATTCCAG CCCAGGTGAA GCTGAGCCCT GAGOCCAGOG
ACCTCATCAC CAAGCTGTGC TGCTCCGCAG ACCACCGCCT GGGGCGGAAT GGGGCCGATG
ACCTGAAGGC CCACCCCTTC TTCAGCGCCA TTGACTTCTC CAGTGACATC CGGAAGCATC
CAGCCCCCTA CGTTCCCACC ATCAGCCACC CCATGGAG. Name: SS1771A Nucleotide
sequence: (SEQ ID NO:15)
TCCCGCTGGACGTGGAGTACGGAGGCCCAGACCGGAGGTGCCCGCCTCCGCCCTACCCGAAGCACCTGCTGC
TGCGCAGCAAGTCGGAGCAGTACGACCTGGACAGCCTGTGCGCAGGCATGGAGCAGAGCCTCCGT-
GCGGGCC
CCAACGAGCCCGAGGGCGGCGACAAGAGCCGCAAAAGCGCCAAGGGGGACAAAGGCGGA-
AAGGATAAAAAGC
AGATTCAGACCTCTCCCGTTCCCGTCCGCAAAAACAGCAGAGACGAAGAGAAG-
AGAGAGTCACGCATCAAGA
GCTACTCGCCATACGCCTTTAAGTTCTTCATGGAGCAGCACGTGGAG-
AATGTCATCAAAACCTACCAGCAGA
AGGTTAACCGGAGGCTGCAGCTGGAGCAAGAAATGGCCAAA-
GCTGGACTCTGTGAAGCTGAGCAGGAGCAGA
TGCGGAAGATCCTCTACCAGAAAGAGTCTAATTAC-
AACAGGTTAAAGAGGGCCAAGATGGACAAGTCTATGT
TTGTCAAGATCAAAACCCTGGGGATCGGT-
GCCTTTGGAGAAGTGTGCCTTGCTTGTAAGGTGGACACTCACG
CCCTGTACGCCATGAAGACCCTA-
AGGAAAAAGGATGTCCTGAACCGGAATCAGGTGGCCCACGTCAAGGCCG
AGAGGGACATCCTGGCCGAGGCAGACAATGAGTGGGTGGTCAAACTCTACTACTCCTTCCAAGACAAAGACA
GCCTGTACTTTGTGATGGACTACATCCCTGGTGGGGACATGATGAGCCTGCTGATCCGGATGGAGG-
TCTTCC
CTGAGCACCTGGCCCGGTTCTACATCGCAGAGCTGACTTTGGCCATTGAGAGTGTCCACA-
AGATGGGCTTCA
TCCACCGAGACATCAAGCCTGATAACATTTTGATAGATCTGGATGGTCACATTA-
AACTCACAGATTTCGGCC
TCTGCACTGGGTTCAGGTGGACTCACAATTCCAAATATTACCAGAAAG-
GGAGCCATGTCAGACAGGACAGCA
TGGAGCCCAGCGACCTCTGGGATGATGTGTCTAACTGTCGGT-
GTGGGGACAGGCTGAAGACCCTAGAGCAGA
GGGCGCGGAAGCAGCACCAGAGGTGCCTGGCACATT-
CACTGGTGGGGACTCCAAACTACATCGCACCCGAGG
TGCTCCTCCGCAAAGGGTACACTCAACTCT-
GTGACTGGTGGAGTGTTGGAGTGATTCTCTTCGAGATGCTGG
TGGGGCAGCCGCCCTTTTTGGCAC-
CTACTCCCACAGAAACCCAGCTGAAGGTGATCAACTGGGAGAACACGC
TCCACATTCCAGCCCAGGTGAAGCTGAGCCCTGAGGCCAGGGACCTCATCACCAAGCTGTGCTGCTCCGCAG
ACCACCGCCTGGGGCGGAATGGGGCCGATGACCTGAAGGCCCACCCCTTCTTCAGCGCCATTGACT-
TCTCCA
GTGACATCCGGAAGCATCCAGCCCCCTACGTTCCCACCATCAGCCACCCCATGGACACCT-
CGAATTTCGACC
CCGTAGATGAAGAAAGCCCTTGGAACGATGCCAGCGAAGGTAGCACCAAGGCCT-
GGGACACACTCACCTCGC
CCAATAACAAGCATCCTGACCACGCATTTTACGAATTCACCTTCCGAA-
GGTTCTTTGATGACAATGGCTACC
CCTTTCGATGCCCAAAGCCTTCAGGAGCAGAAGCTTCACAGG-
CTGAGAGCTCAGATTTAGAAAGCTCTGATC
TGGTGGATCAGACTGAAGGCTGCCAGCCTCTGTACG-
TGTAGATGGGGGCCAGGCACCCCCACCACTCGCTGC
CTCCCAGGTCAGGGTCCCGGAGCCGGTGCC-
CTCACAGGCCAATAGGGAAGCCGAGGGCTGTTTTGTTTTAAA
TTAGTCCGTCGATTACTTCACTTG-
AAATTCTGCTCTTCACCAAGAAAACCCAAACAGGACACTTTTGAAAAC
AGCGGTGCCGCGAATTC.
[0022] With the continued increase in the number of known
eukaryotic protein kinases, a suitable classification scheme is one
based on comparing catalytic-domain sequences. It follows that
protein kinases with similar catalytic domains will tend also to
have similar enzymatic and regulatory properties. The nucleotide
sequences of the invention encode, respectively, the following
polypeptides having kinase function:
[0023] NAME: HH0900-BF04 Polypeptide
[0024] Translation in Relevant Reading Frame (3'-5'Frame 3):
2 (SEQ ID NO:7) YAMKVLRKAALVQRAKTQEHTRTERSVLELVRQAPFLVTLHYA-
FQTDAKL HLILDYVSGG;
[0025] The above sequence is consistent with the consensus sequence
of subdomains II through V of the eukaryotic protein kinase
superfamily. This sequence is the sequence of a polypeptide having
kinase activity encoded by the nucleotide sequence of SEQ ID
NO:1.
[0026] NAME: HH2040-BF04 Polypeptide
[0027] Translation in Relevant Reading Frame (5'-3'Frame 2):
3 (SEQ ID NO:8) REVPHQTASDFVRDSAASHQAEPEAYERRVCFLLLQLCNGLE
HLKEHGIIHRDLCLENLLLVHCTLQAGPGPA;
[0028] The above sequence is consistent with the consensus sequence
of subdomains V, VIA and VIB of the eukaryotic protein kinase
superfamily. This sequence is the sequence of a polypeptide having
kinase activity encoded by the nucleotide sequence of SEQ ID
NO:2.
[0029] Name: JJ503-KS Polypeptide
[0030] Translation in Relevant Reading Frame (5'-3' Frame 1):
4 (SEQ ID NO:9) GQGWSWAGIPSSAAAQRAGPPAGALEALSPGGARAHAERRGEM-
RATPLAA PAGSLSRKKRLELDDNLDTERPVQKRARSGPQPRLPPCLLPLSPPTAPD- R
ATAVATASRLGPYVLLEPEEGGRAYQALHCPTGTEYTCKVYPVQEALAVL
EPYARLPPHKHVARPTEVLAGTQLLYAFFTRTHGDMHSLVRSRHRIPEPE
AAVLFRQMATALAHCHQHGLVLRDLKLCRFVFADRERKKLVLENLEDSCV
LTGPDDSLWDKHACPAYVGPEILSSRASYSGKAADVWSLGVALFTMLAGH
YPFQDSEPVLLFGKIRRGAYALPAGLSAPARCLVRCLLRREPAERLTATG ILLHPWLRQD;
[0031] The sequence is consistent with kinase domains VIA through
XI, though the homology is imperfect. This sequence is the sequence
of a polypeptide having kinase activity encoded by the nucleotide
sequence of SEQ ID NO:3.
[0032] Name: QQ1249-BF04 Polypeptide
[0033] Translation in Relevant Reading Frame (5'-3' Frame 3)
5 (SEQ ID NO:10) QREADMHRLFNHPNILRLVAYCLRERGAKHEAWLLLPFFKRG-
TLWNEIER LKDKGNFLTEDQILWLLLGICRGLEAIHAKGYAYRDLKPTNILLGDEG- QP
VLMDLGSMNQACIHVEGSRQALTLQDWAAQRCTISYRAPXLFSVQS;
[0034] The above sequence is consistent with the consensus sequence
of subdomains III-VIII of the eukaryotic protein kinase
superfamily. This sequence is the sequence of a polypeptide having
kinase activity encoded by the nucleotide sequence of SEQ ID
NO:4.
[0035] Name: QQ3351-BF04 Polypeptide
[0036] Translation in Relevant Frame (5'-3' Frame 1)
6 (SEQ ID NO:11) MLTSLNRSWNETTCCGRASFLELCTGQIGRTPLGRREGMENL-
KHIITLGQ VIHKRCEEMKYCKKQCRRLGHRVLGLIKPLEMLQDQGKRSVPSEKLTT- AM
NRFKAALEEANGEIEKFSNRSNICRFLTASQDKILFKDVNRKLSDVWKEL
SLLLQVEQRMPVSPISQGASWAQEDQQDADEDRRAFQMLRRDNEKIEASL
RRLEINMKEIKETLRQYLPPKCMQEIPQEQIKEIKKEQLSGSPWILLREN
EVSTLYKGEYHRAPVAIKVFKKLQAGSIAIVRQTFNKEIKTMKKFESPNI
LRIFGICIDETVTPPQFSIVMEYCELGTLRELLDREKDLTLGKRMVLVLG
AARGLYRLHHSEAPELHGKIRSSNFLVTQGYQVKLAGFELRKTQTSMSLG
TTREKTDRVKSTAYLSPQELEDVFYQYDVKSEIYSFGIVLWEIATGDIPF
QGCNSEKIRKLVAVKRQQEPLGEDCPSELREIIDECRAAGRLVPRSVAAA RAVDV
[0037] The above sequence is believed to be full-length. However,
the initial methionine was not present in the clone sequenced but
subsequently was added by PCR. Therefore, the natural sequence may
comprise Leu-2 through the Val-505. This sequence is the sequence
of a polypeptide having kinase activity encoded by the nucleotide
sequence of SEQ ID NO:5.
[0038] Name: SS1771
[0039] Translation in Relevant Frame (3'-5' Frame 3)
7 (SEQ ID NO:12) FPLDVEYGGPDRRCPPPPYPKHLLLRSKSEQYDLDSLCAGME-
QSLRAGPN EPEGGDKSRKSAKGDKGGKDKKQIQTSPVPVRKNSRDEEKRESRIKSY- SP
YAFKFFMEQHVENVIKTYQQKVNRRLQLEQEMAKAGLCEAEQEQMRKILY
QKESNYNRLKRAKMDKSMFVKIKTLGIGAFGEVCLACKVDTHALYAMKTL
RKKDVLNRNQVAHVKAERDILAEADNEWVVKLYYSFQDKDSLYFVMDYIP
GGDMMSLLIRMEVFPEHLARFYIAELTLAIESVHKMGFIHRDIKPDNILI
DLDGHIKLTDFGLCTGFRWTHNSKYYQKGSHVRQDSMEPSDLWDDVSNCR
CGDRLKTLEQRARKQHQRCLAHSLVGTPNYIAPEVLLRKGYTQLCDWWSV
GVILFEMLVGQPPFLAPTPTETQLKVINWENTLHIPAQVKLSPEARDLIT
KLCCSADHRLGRNGADDLKAHPFFSAIDFSSDIRKHPAPYVPTISHPME.
[0040] This sequence is the sequence of a polypeptide having kinase
activity encoded by the nucleotide sequence of SEQ ID NO:6. The
sequence may also comprise Pro-2 through Glu-499.
[0041] Name: SS1771A
[0042] Translation in Relevant Frame (5'-3' Frame 3)
8 (SEQ ID NO:16) PLDVEYGGPDRRCPPPPYPKHLLLRSKSEQYDLDSLCAGMEQ-
SLRAGPNE PEGGDKSRKSAKGDKGGKDKKQIQTSPVPVRKNSRDEEKRESRIKSYS- PY
AFKFFMEQHVENVIKTYQQKVNRRLQLEQEMAKAGLCEAEQEQMRKILYQ
KESNYNRLKRAKMDKSMFVKIKTLGIGAFGEVCLACKVDTHALYAMKTLR
KKDVLNRNQVAHVKAERDILAEADNEWVVKLYYSFQDKDSLYFVMDYIPG
GDMMSLLIRMEVFPEHLARFYIAELTLAIESVHKMGFIHRDIKPDNILID
LDGHIKLTDFGLCTGFRWTHNSKYYQKGSHVRQDSMEPSDLWDDVSNCRC
GDRLKTLEQRARKQHQRCLAHSLVCTPNYIAPEVLLRKGYTQLCDWWSVG
VILFEMLVGQPPFLAPTPTETQLKVINWENTLHIPAQVKLSPEARDLITK
LCCSADHRLGRNGADDLKAHPFFSAIDFSSDIRKHPAPYVPTISHPMDTS
NFDPVDEESPWNDASEGSTKAWDTLTSPNNKHPEHAFYEFTFRRFFDDNG
YPFRCPKPSGAEASQAESSDLESSDLVDQTEGCQPVYV
[0043] This sequence is the sequence of a polypeptide having kinase
activity encoded by the nucleotide sequence of SEQ ID NO:1.
[0044] The invention also includes truncated forms of the nucleic
acids and polypeptides of the invention. In a preferred embodiment,
the invention includes a truncated form of QQ3351-BF04, as
follows:
[0045] Nucleotide Sequence:
9 (SEQ ID NO:13) CTTGCAGGATTTGAGTTGAGGAAAACACAGACTTCCATGAGT-
TTGGGAAC TACGAGAGAAAAGACAGACAGAGTCAAATCTACAGCATATCTCTCACC- TC
AGGAACTGGAAGATGTATTTTATCAATATGATGTAAAGTCTGAAATATAC
AGCTTTGGAATCGTCCTCTGGGAAATCGCCACTGGAGATATCCCGTTTCA
AGGCTGTAATTCTGAGAAGATCCGCAAGCTGGTGGCTGTGAAGCGGCAGC
AGGAGCCACTGGGTGAAGACTGCCCTTCAGAGCTGCGGGAGATCATTGAT
GAGTGCCGGGCCCATGATCCCTCTGTGCGGCCCTCTGTGGATGAAATCTT
AAAGAAACTCTCCACCTTTTCTAAG
[0046] Translation in Relevant Frame (5'3' Frame 1):
10 (SEQ ID NO:14) LAGFELRKTQTSMSLGTTREKTDRVKSTAYLSPQELEDVFY-
QYDVKSEIY SFGIVLWEIATGDIPFQGCNSEKIRKLVAVKRQQEPLGEDCPSELRE- IID
ECRAHDPSVRPSVDEILKKLSTFSK
[0047] This sequence is the sequence of a polypeptide having kinase
activity encoded by the nucleotide sequence of SEQ ID NO:13.
[0048] The polypeptides of the invention are useful for
characterizing cell and tissue expression, understanding their
roles in development or hormonal response, and identifying
regulatory molecules and physiologically relevant protein
substrates.
[0049] As used herein, the term "polypeptides of the invention"
refers to a genus of polypeptides that further encompasses proteins
having the amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, 12,
14, or 16 as well as those proteins having a high degree of
similarity (at least 90% homology) with such amino acid sequences
and which proteins are biologically active. In addition,
polypeptides of the invention refers to the gene products of the
nucleotides of SEQ ID NO:1, 2, 3, 4, 5, 6, 13 or 15.
[0050] The isolated and purified polypeptides of the invention have
molecular weights of approximately 6883 (HH0900-BF04); 8168
(HH2040-BF04); 39,284 (JJ503-KS); 16,718 (QQ1249-BF04); 58,001
(QQ3351-BF04); 57,381 (SS1771), and 67,331 (SS1771A) Daltons in the
absence of glycosylation. It is understood that the molecular
weight of these polypeptides can be varied by fusing additional
peptide sequences to both the amino and carboxyl terminal ends of
polypeptides of the invention. Fusions of additional peptide
sequences at the amino and carboxyl terminal ends of polypeptides
of the invention can be used to enhance expression of these
polypeptides or aid in the purification of the protein.
[0051] It is understood that fusions of additional peptide
sequences at the amino and carboxyl terminal ends of polypeptides
of the invention will alter some, but usually not all, of the
fragmented peptides of the polypeptides generated by enzymatic or
chemical treatment.
[0052] It is understood that mutations can be introduced into
polypeptides of the invention using routine and known techniques of
molecular biology. It is further understood that a mutation can be
designed so as to eliminate a site of proteolytic cleavage by a
specific enzyme or a site of cleavage by a specific chemically
induced fragmentation procedure. It is also understood that the
elimination of the site will alter the peptide fingerprint of
polypeptides of the invention upon fragmentation with the specific
enzyme or chemical procedure.
[0053] The term "isolated and purified" as used herein, means that
the polypeptides or fragments of the invention are essentially free
of association with other proteins or polypeptides, for example, as
a purification product of recombinant host cell culture or as a
purified product from a non-recombinant source. The term
"substantially purified" as used herein, refers to a mixture that
contains polypeptides or fragments of the invention and is
essentially free of association with other proteins or
polypeptides, but for the presence of known proteins that can be
removed using a specific antibody, and which substantially purified
polypeptides or fragments thereof can be used as molecular weight
markers. The term "purified" refers to either the "isolated and
purified" form of polypeptides of the invention or the
"substantially purified" form of polypeptides of the invention, as
both are described herein.
[0054] A polypeptide "variant" as referred to herein means a
polypeptide substantially homologous to native polypeptides of the
invention, but which has an amino acid sequence different from that
of native polypeptides (human, murine or other mammalian species)
of the invention because of one or more deletions, insertions or
substitutions. The variant amino acid sequence preferably is at
least 80% identical to a native polypeptide amino acid sequence,
most preferably at least 90% identical. The percent identity can be
determined, for example, by comparing sequence information using
the GAP computer program, version 6.0 described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl.
Math 2:482, 1981). The preferred default parameters for the GAP
program include: (1) a unary comparison matrix (containing a value
of 1 for identities and 0 for non-identities) for nucleotides, and
the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps.
[0055] Variants can comprise conservatively substituted sequences,
meaning that a given amino acid residue is replaced by a residue
having similar physiochemical characteristics.
[0056] Examples of conservative substitutions include substitution
of one aliphatic residue for another, such as Ile, Val, Leu, or Ala
for one another, or substitutions of one polar residue for another,
such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other
such conservative substitutions, for example, substitutions of
entire regions having similar hydrophobicity characteristics, are
well known. Naturally occurring variants are also encompassed by
the invention. Examples of such variants are proteins that result
from alternate mRNA splicing events, proteolytic cleavage of the
polypeptides, or transcription/translation from different alleles.
Variations attributable to proteolysis include, for example,
differences in the N- or C-termini upon expression in different
types of host cells, due to proteolytic removal of one or more
terminal amino acids from the polypeptides (generally from 1-5
terminal amino acids) of the invention.
[0057] The polypeptides of the invention can also exist as
oligomers, such as covalently linked or non-covalently linked
dimers or trimers. Oligomers can be linked by disulfide bonds
formed between cysteine residues on different polypeptides.
[0058] In one embodiment of the invention, a polypeptide dimer is
created by fusing polypeptides of the invention to the Fc region of
an antibody (e.g., IgG1) in a manner that does not interfere with
the biological activity of these polypeptides. The Fc region
preferably is fused to the C-terminus of a soluble polypeptide of
the invention, to form an Fc fusion or an Fc polypeptide. The terms
"Fc fusion protein" or "Fc polypeptides" as used herein includes
native and mutein forms, as well as truncated Fc polypeptides
containing the hinge region that promotes dimerization. Exemplary
methods of making Fc polypeptides set forth above are disclosed in
U.S. Pat. Nos. 5,426,048 and 5,783,672 both of which are
incorporated herein by reference.
[0059] In a preferred embodiment, extracellular domains from
transmembrane bound kinases are fused to Fc portions of antibodies
to produce soluble Fc polypeptides. These constructs can function
as binding sites for the ligand that naturally binds the kinase
receptor and thereby inhibit binding of the ligand to the natural
receptor.
[0060] General preparation of fusion proteins comprising
heterologous polypeptides fused to various portions of
antibody-derived polypeptides (including the Fc domain) has been
described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991) and
Byrn et al. (Nature 344:677, 1990), hereby incorporated by
reference. A gene fusion encoding the polypeptide:Fc fusion protein
of the invention is inserted into an appropriate expression vector.
Polypeptide:Fc fusion proteins are allowed to assemble much like
antibody molecules, whereupon interchain disulfide bonds form
between Fc polypeptides, yielding divalent polypeptides of the
invention. If fusion proteins are made with both heavy and light
chains of an antibody, it is possible to form a polypeptide
oligomer with as many as four polypeptides extracellular regions.
Alternatively, one can link two soluble polypeptide domains with a
peptide linker.
[0061] In one embodiment of this invention, the polypeptides of the
invention are produced by recombinant expression. In one preferred
embodiment, the expression of recombinant polypeptides having
kinase functions can be accomplished utilizing fusion of sequences
encoding polypeptides having kinase functions to sequences encoding
another polypeptide to aid in the purification of polypeptides of
the invention. An example of such a fusion is a fusion of sequences
encoding a polypeptide having kinase functions to sequences
encoding the product of the malE gene of the pMAL-c2 vector of New
England Biolabs, Inc. Such a fusion allows for affinity
purification of the fusion protein, as well as separation of the
maltose binding protein portion of the fusion protein from the
polypeptide of the invention after purification.
[0062] The insertion of DNA encoding the polypeptide having kinase
functions into the pMAL-c2 vector can be accomplished in a variety
of ways using known molecular biology techniques. The preferred
construction of the insertion contains a termination codon
adjoining the carboxyl terminal codon of the polypeptide of the
invention. In addition, the preferred construction of the insertion
results in the fusion of the amino terminus of the polypeptide of
the invention directly to the carboxyl terminus of the Factor Xa
cleavage site in the pMAL-c2 vector. A DNA fragment can be
generated by PCR using DNA of the invention as the template DNA and
two oligonucleotide primers. Use of the oligonucleotide primers
generates a blunt-ended fragment of DNA that can be isolated by
conventional means. This PCR product can be ligated together with
pMAL-p2 (digested with the restriction endonuclease Xmn I) using
conventional means. Positive clones can be identified by
conventional means. Induction of expression and purification of the
fusion protein can be performed as per the manufacturer's
instructions. This construction facilitates a precise separation of
the polypeptide of the invention from the fused maltose binding
protein utilizing a simple protease treatment as per the
manufacturer's instructions. In this manner, purified polypeptide
having kinase functions can be obtained. Furthermore, such a
constructed vector can be easily modified using known molecular
biology techniques to generate additional fusion proteins. It is
understood, of course, that many different vectors and techniques
can be used for the expression and purification of polypeptides of
the invention and that this embodiment in no way limits the scope
of the invention.
[0063] Polypeptides of the invention can be subjected to
fragmentation into peptides by chemical and enzymatic means.
Chemical fragmentation includes the use of cyanogen bromide to
cleave under neutral or acidic conditions such that specific
cleavage occurs at methionine residues (E. Gross, Methods in Enz.
11:238-255, 1967). This can further include additional steps, such
as a carboxymethylation step to convert cysteine residues to an
unreactive species. Enzymatic fragmentation includes the use of a
protease such as Asparaginylendopeptidase, Arginylendo-peptidase,
Achromobacter protease I, Trypsin, Staphlococcus aureus V8
protease, Endoproteinase Asp-N, or Endoproteinase Lys-C under
conventional conditions to result in cleavage at specific amino
acid residues. Asparaginylendo-peptidase can cleave specifically on
the carboxyl side of the asparagine residues present within the
polypeptides of the invention. Arginylendo-peptidase can cleave
specifically on the carboxyl side of the arginine residues present
within these polypeptides. Achromobacter protease I can cleave
specifically on the carboxyl side of the lysine residues present
within the polypeptides (Sakiyama and Nakat, U.S. Pat. No.
5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50,
1981; T. Masaki et al., Biochim. Biophys. Acta 660:51-55, 1981).
Trypsin can cleave specifically on the carboxyl side of the
arginine and lysine residues present within polypeptides of the
invention. Staphlococcus aureus V8 protease can cleave specifically
on the carboxyl side of the aspartic and glutamic acid residues
present within polypeptides (D. W. Cleveland, J. Biol. Chem.
3:1102-1106, 1977). Endoproteinase Asp-N can cleave specifically on
the amino side of the asparagine residues present within
polypeptides. Endoproteinase Lys-C can cleave specifically on the
carboxyl side of the lysine residues present within polypeptides of
the invention. Other enzymatic and chemical treatments can likewise
be used to specifically fragment these polypeptides into a unique
set of specific peptide molecular weight markers.
[0064] The resultant fragmented peptides can be analyzed by methods
including sedimentation, electrophoresis, chromatograpy, and mass
spectrometry. The fragmented peptides derived from the polypeptides
of the invention can serve as molecular weight markers using such
analysis techniques to assist in the determination of the molecular
weight of a sample protein. Such a molecular weight determination
assists in the identification of the sample protein. Fragmented
peptide molecular weight markers of the invention are preferably at
least 10 amino acids in size. More preferably, these fragmented
peptide molecular weight markers are between 10 and 100 amino acids
in size. Even more preferable are fragmented peptide molecular
weight markers between 10 and 50 amino acids in size and especially
between 10 and 35 amino acids in size. Most preferable are
fragmented peptide molecular weight markers between 10 and 20 amino
acids in size.
[0065] Furthermore, analysis of the progressive fragmentation of
the polypeptides of the invention into specific peptides (D. W.
Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977), such as by
altering the time or temperature of the fragmentation reaction, can
be used as a control for the extent of cleavage of a sample
protein. For example, cleavage of the same amount of polypeptide
and sample protein under identical conditions can allow for a
direct comparison of the extent of fragmentation. Conditions that
result in the complete fragmentation of the polypeptide can also
result in complete fragmentation of the sample protein.
[0066] In addition, the polypeptides and fragmented peptides of the
invention possess unique charge characteristics and, therefore, can
serve as specific markers to assist in the determination of the
isoelectric point of a sample protein or fragmented peptide using
techniques such as isoelectric focusing. The technique of
isoelectric focusing can be further combined with other techniques
such as gel electrophoresis to simultaneously separate a protein on
the basis of molecular weight and charge. An example of such a
combination is that of two-dimensional electrophoresis (T. D. Brock
and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall,
6d ed. 1991)). These polypeptides and fragmented peptides thereof
can be used in such analyses as markers to assist in the
determination of both the isoelectric point and molecular weight of
a sample protein or fragmented peptide.
[0067] Kits to aid in the determination of apparent molecular
weight and isoelectric point of a sample protein can be assembled
from the polypeptides and peptide fragments of the invention. Kits
also serve to assess the degree of fragmentation of a sample
protein. The constituents of such kits can be varied, but typically
contain the polypeptide and fragmented peptide molecular weight
markers. Also, such kits can contain the polypeptides wherein a
site necessary for fragmentation has been removed. Furthermore, the
kits can contain reagents for the specific cleavage of the
polypeptide and the sample protein by chemical or enzymatic
cleavage. Kits can further contain antibodies directed against
polypeptides or fragments thereof of the invention.
[0068] The isolated and purified polypeptides of the invention have
molecular weights of approximately 6883 (HH0900-BF04); 8168
(HH2040-BF04); 39,284 (JJ503-KS); 16,718 (QQ1249-BF04); 58,001
(QQ3351-BF04); 57,381 (SS1771), and 67,331 (SS1771A) Daltons in the
absence of glycosylation. The polypeptide of the invention,
together with a sample protein, can be resolved by denaturing
polyacrylamide gel electrophoresis by conventional means (U. K.
Laemmli, Nature 227:680-685, 1970) in two separate lanes of a gel
containing sodium dodecyl sulfate and a concentration of acrylamide
between 6-20%. Proteins on the gel can be visualized using a
conventional staining procedure. The polypeptide molecular weight
markers of the invention can be used as molecular weight marker in
the estimation of the apparent molecular weight of the sample
protein. The unique amino acid sequence of SEQ ID NO:7, 8, 9, 10,
11, 12, and 16 correspond to molecular weight of approximately
6883; 8168; 39,284; 16,718; 58,001; 57,381; or 67,331 Daltons,
respectively. Therefore, the polypeptide molecular weight markers
serve particularly well as a molecular weight marker for the
estimation of the apparent molecular weight of sample proteins that
have apparent molecular weights close to 6883; 8168; 39,284;
16,718; 58,001; 57,381; or 67,331 Daltons. The use of these
polypeptide molecular weight markers allows increased accuracy in
the determination of apparent molecular weight of proteins that
have apparent molecular weights close to 6883; 8168; 39,284;
16,718; 28,982; or 57,381 Daltons. It is understood of course that
many different techniques can be used for the determination of the
molecular weight of a sample protein using polypeptides of the
invention, and that this embodiment in no way limits the scope of
the invention.
[0069] Another preferred embodiment of the invention is the use of
polypeptides and fragmented peptides of the invention as molecular
weight markers to estimate the apparent molecular weight of a
sample protein by gel electrophoresis. These fragmented peptides
can be generated methods well known in the art such as chemical
fragmentation. Isolated and purified polypeptides of the invention
can be treated with cyanogen bromide under conventional conditions
that result in fragmentation of the polypeptide molecular weight
marker by specific hydrolysis on the carboxyl side of the
methionine residues within the polypeptides of the invention (E.
Gross, Methods in Enz. 11:238-255, 1967). Due to the unique amino
acid sequence of the polypeptides of the invention, the
fragmentation of polypeptide molecular weight markers with cyanogen
bromide generates a unique set of fragmented peptide molecular
weight markers. The distribution of methionine residues determines
the number of amino acids in each peptide and the unique amino acid
composition of each peptide determines its molecular weight.
Polypeptide molecular weight markers of the invention can be
analyzed by methods including sedimentation, gel electrophoresis,
chromatography, and mass spectrometry.
[0070] The fragmented peptide molecular weight markers of the
invention, together with a sample protein, can be resolved by
denaturing polyacrylamide gel electrophoresis by conventional means
in two separate lanes of a gel containing sodium dodecyl sulfate
and a concentration of acrylamide between 10-20%. Proteins on the
gel can be visualized using a conventional staining procedure. The
fragmented peptide molecular weight markers of the invention can be
used as molecular weight markers in the estimation of the apparent
molecular weight of the sample protein. The unique amino acid
sequence of each marker specifies a molecular weight. Therefore,
the fragmented peptide molecular weight markers serve particularly
well as molecular weight markers for the estimation of the apparent
molecular weight of sample proteins that have similar apparent
molecular weights. Consequently, the use of these fragmented
peptide molecular weight markers allows increased accuracy in the
determination of apparent molecular weight of proteins.
[0071] Polypeptides on the membrane can be visualized using two
different methods that allow a discrimination between the sample
protein and the molecular weight markers. Polypeptide or fragmented
peptide molecular weight markers of the invention can be visualized
using antibodies generated against these markers and conventional
immunoblotting techniques. This detection is performed under
conventional conditions that do not result in the detection of the
sample protein. It is understood that it may not be possible to
generate antibodies against all polypeptide fragments of the
invention, since small peptides may not contain immunogenic
epitopes. It is further understood that not all antibodies will
work in this assay; however, those antibodies which are able to
bind polypeptides and fragments of the invention can be readily
determined using conventional techniques.
[0072] The sample protein is visualized using a conventional
staining procedure. The molar excess of sample protein to
polypeptide or fragmented peptide molecular weight markers of the
invention is such that the conventional staining procedure
predominantly detects the sample protein. The level of these
polypeptide or fragmented peptide molecular weight markers is such
as to allow little or no detection of these markers by the
conventional staining method. The preferred molar excess of sample
protein to polypeptide molecular weight markers of the invention is
between 2 and 100,000 fold. More preferably, the preferred molar
excess of sample protein to these polypeptide molecular weight
markers is between 10 and 10,000 fold and especially between 100
and 1,000 fold.
[0073] The polypeptide or fragmented peptide molecular weight
markers of the invention can be used as molecular weight and
isoelectric point markers in the estimation of the apparent
molecular weight and isoelectric point of the sample protein. These
polypeptide or fragmented peptide molecular weight markers serve
particularly well as molecular weight and isoelectric point markers
for the estimation of apparent molecular weights and isoelectric
points of sample proteins that have apparent molecular weights and
isoelectric points close to that of the polypeptide or fragmented
peptide molecular weight markers of the invention. The ability to
simultaneously resolve these polypeptide or fragmented peptide
molecular weight markers and the sample protein under identical
conditions allows for increased accuracy in the determination of
the apparent molecular weight and isoelectric point of the sample
protein. This is of particular interest in techniques, such as two
dimensional electrophoresis, where the nature of the procedure
dictates that any markers should be resolved simultaneously with
the sample protein.
[0074] In another embodiment, polypeptide or fragmented peptide
molecular weight markers of the invention can be used as molecular
weight and isoelectric point markers in the estimation of the
apparent molecular weight and isoelectric point of fragmented
peptides derived by treatment of a sample protein with a cleavage
agent. It is understood of course that many techniques can be used
for the determination of molecular weight and isoelectric point of
a sample protein and fragmented peptides thereof using these
polypeptide molecular weight markers and peptide fragments thereof
and that this embodiment in no way limits the scope of the
invention.
[0075] Polypeptide molecular weight markers encompassed by
invention can have variable molecular weights, depending upon the
host cell in which they are expressed. Glycosylation of polypeptide
molecular weight markers and peptide fragments of the invention in
various cell types can result in variations of the molecular weight
of these markers, depending upon the extent of modification. The
size of these polypeptide molecular weight markers can be most
heterogeneous with fragments of polypeptide derived from the
extracellular portion of the polypeptide. Consistent molecular
weight markers can be obtained by using polypeptides derived
entirely from the transmembrane and cytoplasmic regions,
pretreating with N-glycanase to remove glycosylation, or expressing
the polypeptides in bacterial hosts.
[0076] As stated above, the invention provides isolated and
purified polypeptides, both recombinant and non-recombinant.
Variants and derivatives of native polypeptides can be obtained by
mutations of nucleotide sequences coding for native polypeptides.
Alterations of the native amino acid sequence can be accomplished
by any of a number of conventional methods. Mutations can be
introduced at particular loci by synthesizing oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes an analog having the
desired amino acid insertion, substitution, or deletion.
[0077] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
wherein predetermined codons can be altered by substitution,
deletion or insertion. Exemplary methods of making the alterations
set forth above are disclosed by Walder et al. (Gene 42:133, 1986);
Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January
1985, 12-19); Smith et al. (Genetic Engineering: Principles and
Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA
82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987);
and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of which are
incorporated by reference.
[0078] Polypeptides of the invention can be modified to create
polypeptide derivatives by forming covalent or aggregative
conjugates with other chemical moieties, such as glycosyl groups,
polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups
and the like. Covalent derivatives of polypeptides of the invention
can be prepared by linking the chemical moieties to functional
groups on polypeptide amino acid side chains or at the N-terminus
or C-terminus of a polypeptide of the invention or the
extracellular domain thereof. Other derivatives of polypeptides
within the scope of this invention include covalent or aggregative
conjugates of these polypeptides or peptide fragments with other
proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions. For example, the
conjugate can comprise a signal or leader polypeptide sequence
(e.g. the .alpha.-factor leader of Saccharomyces) at the N-terminus
of a polypeptide of the invention. The signal or leader peptide
co-translationally or post-translationally directs transfer of the
conjugate from its site of synthesis to a site inside or outside of
the cell membrane or cell wall.
[0079] Polypeptide conjugates can comprise peptides added to
facilitate purification and identification of polypeptides of the
invention. Such peptides include, for example, poly-His or the
antigenic identification peptides described in U.S. Pat. No.
5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.
[0080] The invention further includes polypeptides of the invention
with or without associated native-pattern glycosylation.
Polypeptides expressed in yeast or mammalian expression systems
(e.g., COS-1 or COS-7 cells) can be similar to or significantly
different from a native polypeptide in molecular weight and
glycosylation pattern, depending upon the choice of expression
system. Expression of polypeptides of the invention in bacterial
expression systems, such as E. coli, provides non-glycosylated
molecules. Glycosyl groups can be removed through conventional
methods, in particular those utilizing glycopeptidase. In general,
glycosylated polypeptides of the invention can be incubated with a
molar excess of glycopeptidase (Boehringer Mannheim).
[0081] Correspondingly, equivalent DNA constructs that encode
various additions or substitutions of amino acid residues or
sequences, or deletions of terminal or internal residues or
sequences are encompassed by the invention. For example,
N-glycosylation sites in the polypeptide extracellular domain can
be modified to preclude glycosylation, allowing expression of a
reduced carbohydrate analog in mammalian and yeast expression
systems. N-glycosylation sites in eukaryotic polypeptides are
characterized by an amino acid triplet Asn-X-Y, wherein X is any
amino acid except Pro and Y is Ser or Thr. Appropriate
substitutions, additions, or deletions to the nucleotide sequence
encoding these triplets will result in prevention of attachment of
carbohydrate residues at the Asn side chain. Alteration of a single
nucleotide, chosen so that Asn is replaced by a different amino
acid, for example, is sufficient to inactivate an N-glycosylation
site. Known procedures for inactivating N-glycosylation sites in
proteins include those described in U.S. Pat. No. 5,071,972 and EP
276,846, hereby incorporated by reference.
[0082] In another example, sequences encoding Cys residues that are
not essential for biological activity can be altered to cause the
Cys residues to be deleted or replaced with other amino acids,
preventing formation of incorrect intramolecular disulfide bridges
upon renaturation. Other equivalents are prepared by modification
of adjacent dibasic amino acid residues to enhance expression in
yeast systems in which KEX2 protease activity is present. EP
212,914 discloses the use of site-specific mutagenesis to
inactivate KEX2 protease processing sites in a protein. KEX2
protease processing sites are inactivated by deleting, adding, or
substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs
to eliminate the occurrence of these adjacent basic residues.
Lys-Lys pairings are considerably less susceptible to KEX2
cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys
represents a conservative and preferred approach to inactivating
KEX2 sites.
[0083] The invention further encompasses isolated fragments and
oligonucleotides derived from the nucleotide sequence of SEQ ID
NO:1, 2, 3, 4, 5, 6, 13 or 15. Nucleic acid sequences within the
scope of the invention include isolated DNA and RNA sequences that
hybridize to the native nucleotide sequences disclosed herein under
conditions of moderate or severe stringency, and which encode
polypeptides or fragments thereof of the invention. These isolated
DNA and RNA sequences also include full length DNA or RNA molecules
encoding for polypeptides with kinase activity. As used herein,
conditions of moderate stringency, as known to those having
ordinary skill in the art, and as defined by Sambrook et al.
Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp.
1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use
of a prewashing solution for the nitrocellulose filters
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization
conditions of 50% formamide, 6.times.SSC at 42.degree. C. (or other
similar hybridization solution, such as Stark's solution, in 50%
formamide at 42.degree. C.), and washing conditions of about
60.degree. C., 0.5.times.SSC, 0.1% SDS. Conditions of high
stringency are defined as hybridization conditions as above, and
with washing at 68.degree. C., 0.2.times.SSC, 0.1% SDS. The skilled
artisan will recognize that the temperature and wash solution salt
concentration can be adjusted as necessary according to factors
such as the length of the probe.
[0084] Due to the known degeneracy of the genetic code, wherein
more than one codon can encode the same amino acid, a DNA sequence
can vary from that shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 13 or 15,
and still encode a polypeptide having the amino acid sequence of
SEQ ID NO:7, 8, 9, 10, 11, 12, 14 or 16. Such variant DNA sequences
can result from silent mutations (e.g., occurring during PCR
amplification), or can be the product of deliberate mutagenesis of
a native sequence.
[0085] The invention thus provides equivalent isolated DNA
sequences encoding polypeptides of the invention, selected from:
(a) DNA derived from the coding region of a native mammalian gene;
(b) cDNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 3,
4, 5, 6, 13 or 15; (c) DNA encoding the polypeptides of SEQ ID
NO:7, 8, 9, 10, 11, 12, 14 or 16; (d) DNA capable of hybridization
to a DNA of (a) under conditions of moderate stringency and which
encodes polypeptides of the invention; and (e) DNA which is
degenerate as a result of the genetic code to a DNA defined in (a),
(b), (c), or (d) and which encodes polypeptides of the invention.
Of course, polypeptides encoded by such DNA equivalent sequences
are encompassed by the invention.
[0086] DNA that is equivalent to the DNA sequence of SEQ ID NO:1,
2, 3, 4, 5, 6, 13 or 15 will hybridize under moderately stringent
conditions to the double-stranded native DNA sequence that encode
polypeptides comprising amino acid sequences of SEQ ID NO:7, 8, 9,
10, 11, 12, 14 or 16. Examples of polypeptides encoded by such DNA,
include, but are not limited to, polypeptide fragments and
polypeptides comprising inactivated N-glycosylation site(s),
inactivated protease processing site(s), or conservative amino acid
substitution(s), as described above. Polypeptides encoded by DNA
derived from other mammalian species, wherein the DNA will
hybridize to the complement of the DNA of SEQ ID NO:1, 2, 3, 4, 5,
6, 13 or 15 are also encompassed.
[0087] Recombinant expression vectors containing a nucleic acid
sequence encoding polypeptides of the invention can be prepared
using well known methods. The expression vectors include a DNA
sequence of the invention operably linked to suitable
transcriptional or translational regulatory nucleotide sequences,
such as those derived from a mammalian, microbial, viral, or insect
gene. Examples of regulatory sequences include transcriptional
promoters, operators, or enhancers, an mRNA ribosomal binding site,
and appropriate sequences which control transcription and
translation initiation and termination. Nucleotide sequences are
"operably linked" when the regulatory sequence functionally relates
to the DNA sequence of the invention. Thus, a promoter nucleotide
sequence is operably linked to a DNA sequence if the promoter
nucleotide sequence controls the transcription of the DNA sequence
of the invention. The ability to replicate in the desired host
cells, usually conferred by an origin of replication, and a
selection gene by which transformants are identified can
additionally be incorporated into the expression vector.
[0088] In addition, sequences encoding appropriate signal peptides
that are not naturally associated with polypeptides of the
invention can be incorporated into expression vectors. For example,
a DNA sequence for a signal peptide (secretory leader) can be fused
in-frame to the nucleotide sequence of the invention so that the
polypeptide is initially translated as a fusion protein comprising
the signal peptide. A signal peptide that is functional in the
intended host cells enhances extracellular secretion of the
polypeptide. The signal peptide can be cleaved from the polypeptide
upon secretion of polypeptide from the cell.
[0089] Suitable host cells for expression of polypeptides of the
invention include prokaryotes, yeast or higher eukaryotic cells.
Appropriate cloning and expression vectors for use with bacterial,
fungal, yeast, and mammalian cellular hosts are described, for
example, in Pouwels et al. Cloning Vectors: A Laboratory Manual,
Elsevier, New York, (1985). Cell-free translation systems could
also be employed to produce polypeptides of the invention using
RNAs derived from DNA constructs disclosed herein.
[0090] Prokaryotes include gram negative or gram positive
organisms, for example, E. coli or Bacilli. Suitable prokaryotic
host cells for transformation include, for example, E. coli,
Bacillus subtilis, Salmonella typhimurium, and various other
species within the genera Pseudomonas, Streptomyces, and
Staphylococcus. In a prokaryotic host cell, such as E. coli, a
polypeptide of the invention can include an N-terminal methionine
residue to facilitate expression of the recombinant polypeptide in
the prokaryotic host cell. The N-terminal Met can be cleaved from
the expressed recombinant polypeptide.
[0091] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. To construct an expression vector using pBR322,
an appropriate promoter and a DNA sequence of the invention are
inserted into the pBR322 vector. Other commercially available
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
Other commercially available vectors include those that are
specifically designed for the expression of proteins; these would
include pMAL-p2 and pMAL-c2 vectors that are used for the
expression of proteins fused to maltose binding protein (New
England Biolabs, Beverly, Mass., USA).
[0092] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature
275:615, 1978; and Goeddel et al., Nature 281:544, 1979),
tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057, 1980; and EP-A-36776), and tac promoter (Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, p. 412, 1982). A particularly useful prokaryotic host
cell expression system employs a phage .lambda.P.sub.L promoter and
a cI857ts thermolabile repressor sequence. Plasmid vectors
available from the American Type Culture Collection, which
incorporate derivatives of the .lambda.P.sub.L promoter, include
plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and
pPLc28 (resident in E. coli RR1 (ATCC 53082)).
[0093] DNA of the invention can be cloned in-frame into the
multiple cloning site of an ordinary bacterial expression vector.
Ideally the vector contains an inducible promoter upstream of the
cloning site, such that addition of an inducer leads to high-level
production of the recombinant protein at a time of the
investigator's choosing. For some proteins, expression levels can
be boosted by incorporation of codons encoding a fusion partner
(such as hexahistidine) between the promoter and the gene of
interest. The resulting "expression plasmid" can be propagated in a
variety of prokaryotic hosts such as E. coli.
[0094] For expression of the recombinant protein, the bacterial
cells are propagated in growth medium until reaching a
pre-determined optical density. Expression of the recombinant
protein is then induced, e.g. by addition of IPTG
(isopropyl-b-D-thiogalactopyranoside), which activates expression
of proteins from plasmids containing a lac operator/promoter. After
induction (typically for 1-4 hours), the cells are harvested by
pelleting in a centrifuge, e.g. at 5,000.times.G for 20 minutes at
4.degree. C.
[0095] For recovery of the expressed protein, the pelleted cells
may be resuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl
and then passed two or three times through a French press. Most
highly-expressed recombinant proteins form insoluble aggregates
known as inclusion bodies. Inclusion bodies can be purified away
from the soluble proteins by pelleting in a centrifuge at
5,000.times.G for 20 minutes, 4.degree. C. The inclusion body
pellet is washed with 50 mM Tris-HCl (pH 8)/1% Triton X-100 and
then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT. Any
material that cannot be dissolved is removed by centrifugation
(10,000.times.G for 20 minutes, 20.degree. C.). The protein of
interest will, in most cases, be the most abundant protein in the
resulting clarified supernatant. This protein may be "refolded"
into the active conformation by dialysis against 50 mM Tris-HCl (pH
8)/5 mM CaCl.sub.2/5 mM Zn(OAc).sub.2/1 mM GSSG/0.1 mM GSH. After
refolding, purification can be carried out by a variety of
chromatographic methods such as ion exchange or gel filtration. In
some protocols, initial purification may be carried out before
refolding. As an example, hexahistidine-tagged fusion proteins may
be partially purified on immobilized nickel.
[0096] While the preceding purification and refolding procedure
assumes that the protein is best recovered from inclusion bodies,
those skilled in the art of protein purification will appreciate
that many recombinant proteins are best purified out of the soluble
fraction of cell lysates. In these cases, refolding is often not
required, and purification by standard chromatographic methods can
be carried out directly.
[0097] Polypeptides of the invention alternatively can be expressed
in yeast host cells, preferably from the Saccharomyces genus (e.g.,
S. cerevisiae). Other genera of yeast, such as Pichia, K. lactis,
or Kluyveromyces, can also be employed. Yeast vectors will often
contain an origin of replication sequence from a 2.mu. yeast
plasmid, an autonomously replicating sequence (ARS), a promoter
region, sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Suitable promoter
sequences for yeast vectors include, among others, promoters for
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Biol. Chem. 255:2073, 1980), or other glycolytic enzymes (Hess et
al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EPA-73,657 or in Fleer et. al.,
Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology,
8:135-139 (1990). Another alternative is the glucose-repressible
ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674,
1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors
replicable in both yeast and E. coli can be constructed by
inserting DNA sequences from pBR322 for selection and replication
in E. coli (Ampr gene and origin of replication) into the
above-described yeast vectors.
[0098] The yeast .alpha.-factor leader sequence can be employed to
direct secretion of a polypeptide of the invention. The
.alpha.-factor leader sequence is often inserted between the
promoter sequence and the structural gene sequence. See, e.g.,
Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad.
Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.
Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence can be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0099] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 101g/ml adenine, and 20 .mu.g/ml
uracil.
[0100] Yeast host cells transformed by vectors containing ADH2
promoter sequence can be grown for inducing expression in a "rich"
medium. An example of a rich medium is one consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml
adenine and 80 .mu.g/ml uracil. Derepression of the ADH2 promoter
occurs when glucose is exhausted from the medium.
[0101] Mammalian or insect host cell culture systems could also be
employed to express recombinant polypeptides of the invention.
Baculovirus systems for production of heterologous proteins in
insect cells are reviewed by Luckow and Summers, Bio/Technology
6:47 (1988). Established cell lines of mammalian origin also can be
employed. Examples of suitable mammalian host cell lines include
the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et
al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL
163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC
CRL 10) cell lines, and the CV-1/EBNA-1 cell line (ATCC CRL 10478)
derived from the African green monkey kidney cell line CVI (ATCC
CCL 70) as described by McMahan et al. (EMBO J. 10: 2821,
1991).
[0102] Established methods for introducing DNA into mammalian cells
have been described (Kaufinan, R. J., Large Scale Mammalian Cell
Culture, 1990, pp. 15-69). Additional protocols using commercially
available reagents, such as Lipofectamine (Gibco/BRL) or
Lipofectamine-Plus, can be used to transfect cells (Felgner et al.,
Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). In addition,
electroporation can be used to transfect mammalian cells using
conventional procedures, such as those in Sambrook et al. Molecular
Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor
Laboratory Press, 1989). Selection of stable transformants can be
performed using methods known in the art, such as, for example,
resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology
185:487-511, 1990, describes several selection schemes, such as
dihydrofolate reductase (DHFR) resistance. A suitable host strain
for DHFR selection can be CHO strain DX-B11, which is deficient in
DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220,
1980). A plasmid expressing the DHFR cDNA can be introduced into
strain DX-B 11, and only cells that contain the plasmid can grow in
the appropriate selective media. Other examples of selectable
markers that can be incorporated into an expression vector include
cDNAs conferring resistance to antibiotics, such as G418 and
hygromycin B. Cells harboring the vector can be selected on the
basis of resistance to these compounds.
[0103] Transcriptional and translational control sequences for
mammalian host cell expression vectors can be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from polyoma virus, adenovirus 2, simian virus 40
(SV40), and human cytomegalovirus. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites can be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained
from a viral genome as a fragment, which can also contain a viral
origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman,
Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can
also be used, provided the approximately 250 bp sequence extending
from the Hind III site toward the Bgl I site located in the SV40
viral origin of replication site is included.
[0104] Additional control sequences shown to improve expression of
heterologous genes from mammalian expression vectors include such
elements as the expression augmenting sequence element (EASE)
derived from CHO cells (Morris et al., Animal Cell Technology,
1997, pp. 529-534) and the tripartite leader (TPL) and VA gene RNAs
from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491,
1982). The internal ribosome entry site (IRES) sequences of viral
origin allows dicistronic mRNAs to be translated efficiently (Oh
and Sarnow, Current Opinion in Genetics and Development 3:295-300,
1993; Ramesh et al., Nucleic Acids Research 24:2697-2700, 1996).
Expression of a heterologous cDNA as part of a dicistronic mRNA
followed by the gene for a selectable marker (e.g. DHFR) has been
shown to improve transfectability of the host and expression of the
heterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplary
expression vectors that employ dicistronic mRNAs are pTR-DC/GFP
described by Mosser et al., Biotechniques 22:150-161, 1997, and
p2A5I described by Morris et al., Animal Cell Technology, 1997, pp.
529-534.
[0105] A useful high expression vector, pCAVNOT, has been described
by Mosley et al., Cell 59:335-348, 1989. Other expression vectors
for use in mammalian host cells can be constructed as disclosed by
Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system
for stable high level expression of mammalian cDNAs in C 127 murine
mammary epithelial cells can be constructed substantially as
described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful
high expression vector, PMLSV N1/N4, described by Cosman et al.,
Nature 312:768, 1984, has been deposited as ATCC 39890. Additional
useful mammalian expression vectors are described in EP-A-0367566,
and in U.S. patent application Ser. No. 07/701,415, filed May 16,
1991, incorporated by reference herein. The vectors can be derived
from retroviruses. In place of the native signal sequence, a
heterologous signal sequence can be added, such as the signal
sequence for IL-7 described in U.S. Pat. No. 4,965,195; the signal
sequence for IL-2 receptor described in Cosman et al., Nature
312:768 (1984); the IL-4 signal peptide described in EP 367,566;
the type I IL-1 receptor signal peptide described in U.S. Pat. No.
4,968,607; and the type II IL-1 receptor signal peptide described
in EP 460,846. Another useful expression vector, pFLAG, can be
used. FLAG technology is centered on the fusion of a low molecular
weight (1 kD), hydrophilic, FLAG marker peptide to the N-Terminus
of a recombinants protein expressed by the pFLAG-1.TM. Expression
Vector (1) (obtained from IBI Kodak).
[0106] An isolated and purified polypeptide according to the
invention can be produced by recombinant expression systems as
described above or purified from naturally occurring cells.
Polypeptides can be substantially purified, as indicated by a
single protein band upon analysis by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE).
[0107] One process for producing polypeptides of the invention
comprises culturing a host cell transformed with an expression
vector comprising a DNA sequence that encodes a polypeptide of the
invention under conditions sufficient to promote expression of the
polypeptide. The polypeptide is then recovered from culture medium
or cell extracts, depending upon the expression system employed. As
is known to the skilled artisan, procedures for purifying a
recombinant protein will vary according to such factors as the type
of host cells employed and whether or not the recombinant protein
is secreted into the culture medium. For example, when expression
systems that secrete the recombinant protein are employed, the
culture medium first can be concentrated using a commercially
available protein concentration filter, for example, an Amicon or
Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a
purification matrix such as a gel filtration medium. Alternatively,
an anion exchange resin can be employed, for example, a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Alternatively, a
cation exchange step can be employed. Suitable cation exchangers
include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are preferred. Finally,
one or more reversed-phase high performance liquid chromatography
(RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica
gel having pendant methyl or other aliphatic groups) can be
employed to further purify the polypeptides. Some or all of the
foregoing purification steps, in various combinations, are well
known and can be employed to provide an isolated and purified
recombinant protein.
[0108] It is possible to utilize an affinity column comprising a
polypeptide-binding protein of the invention, such as a monoclonal
antibody generated against polypeptides of the invention, to
affinity-purify expressed polypeptides. These polypeptides can be
removed from an affinity column using conventional techniques,
e.g., in a high salt elution buffer and then dialyzed into a lower
salt buffer for use or by changing pH or other components depending
on the affinity matrix utilized.
[0109] In this aspect of the invention, polypeptide-binding
proteins, such as the anti-polypeptide antibodies of the invention,
can be bound to a solid phase such as a column chromatography
matrix or a similar substrate suitable for identifying, separating,
or purifying cells that express polypeptides of the invention on
their surface. Adherence of polypeptide-binding proteins of the
invention to a solid phase contacting surface can be accomplished
by any means, for example, magnetic microspheres can be coated with
these polypeptide-binding proteins and held in the incubation
vessel through a magnetic field. Suspensions of cell mixtures are
contacted with the solid phase that has such polypeptide-binding
proteins thereon. Cells having polypeptides of the invention on
their surface bind to the fixed polypeptide-binding protein and
unbound cells then are washed away. This affinity-binding method is
useful for purifying, screening, or separating such
polypeptide-expressing cells from solution. Methods of releasing
positively selected cells from the solid phase are known in the art
and encompass, for example, the use of enzymes. Such enzymes are
preferably non-toxic and non-injurious to the cells and are
preferably directed to cleaving the cell-surface binding
partner.
[0110] Alternatively, mixtures of cells suspected of containing
polypeptide-expressing cells of the invention first can be
incubated with a biotinylated polypeptide-binding protein of the
invention. Incubation periods are typically at least one hour in
duration to ensure sufficient binding to polypeptides of the
invention. The resulting mixture then is passed through a column
packed with avidin-coated beads, whereby the high affinity of
biotin for avidin provides the binding of the polypeptide-binding
cells to the beads. Use of avidin-coated beads is known in the art.
See Berenson, et al. J. Cell. Biochem., 10D:239 (1986). Wash of
unbound material and the release of the bound cells is performed
using conventional methods.
[0111] In the methods described above, suitable polypeptide-binding
proteins are anti-polypeptide antibodies, and other proteins that
are capable of high-affinity binding of polypeptides of the
invention. A preferred polypeptide-binding protein is an
polypeptide monoclonal antibody.
[0112] Recombinant protein produced in bacterial culture is usually
isolated by initial disruption of the host cells, centrifugation,
extraction from cell pellets if an insoluble polypeptide, or from
the supernatant fluid if a soluble polypeptide, followed by one or
more concentration, salting-out, ion exchange, affinity
purification or size exclusion chromatography steps. Finally,
RP-HPLC can be employed for final purification steps. Microbial
cells can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0113] Transformed yeast host cells are preferably employed to
express polypeptides of the invention as a secreted polypeptide in
order to simplify purification. Secreted recombinant polypeptide
from a yeast host cell fermentation can be purified by methods
analogous to those disclosed by Urdal et al. (J. Chromatog.
296:171, 1984). Urdal et al. describe two sequential,
reversed-phase HPLC steps for purification of recombinant human
IL-2 on a preparative HPLC column.
[0114] In yet another embodiment of the invention, antisense or
sense oligonucleotides comprising a single-stranded nucleic acid
sequence (either RNA or DNA) capable of binding to a target mRNA
sequence (forming a duplex) or to the sequence in the
double-stranded DNA helix (forming a triple helix) can be made
according to the invention. Antisense or sense oligonucleotides,
according to the present invention, comprise a fragment of the
coding region of cDNA (SEQ ID NO:1, 2, 3, 4, 5, 6, 13 or 15). Such
a fragment generally comprises at least about 14 nucleotides,
preferably from about 14 to about 30 nucleotides. The ability to
create an antisense or a sense oligonucleotide, based upon a cDNA
sequence for a given protein is described in, for example, Stein
and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,
BioTechniques 6:958, 1988.
[0115] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of complexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means. The
antisense oligonucleotides thus can be used to block expression of
polypeptides of the invention. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation), but retain sequence specificity
to be able to bind to target nucleotide sequences. Other examples
of sense or antisense oligonucleotides include those
oligonucleotides that are covalently linked to organic moieties,
such as those described in WO 90/10448, and other moieties that
increase affinity of the oligonucleotide for a target nucleic acid
sequence, such as poly-(L-lysine). Further still, intercalating
agents, such as ellipticine, and alkylating agents or metal
complexes can be attached to sense or antisense oligonucleotides to
modify binding specificities of the antisense or sense
oligonucleotide for the target nucleotide sequence.
[0116] Antisense or sense oligonucleotides can be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by insertion of the antisense or sense
oligonucleotide into a suitable retroviral vector, then contacting
the cell with the retrovirus vector containing the inserted
sequence, either in vivo or ex vivo. Suitable retroviral vectors
include, but are not limited to, the murine retrovirus M-MuLV, N2
(a retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see PCT Application US
90/02656).
[0117] Alternatively, sense or antisense oligonucleotides also can
be introduced into a cell containing the target nucleotide sequence
by formation of a conjugate with a ligand binding molecule, as
described in WO 91/04753. Suitable ligand binding molecules
include, but are not limited to, cell surface receptors, growth
factors, other cytokines, or other ligands that bind to cell
surface receptors. Preferably, conjugation of the ligand binding
molecule does not substantially interfere with the ability of the
ligand binding molecule to bind to its corresponding molecule or
receptor, or block entry of the sense or antisense oligonucleotide
or its conjugated version into the cell.
[0118] In yet another embodiment, a sense or an antisense
oligonucleotide can be introduced into a cell containing the target
nucleic acid sequence by formation of an oligonucleotide-lipid
complex, as described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0119] Another embodiment of the invention relates to therapeutic
uses of kinases. Kinases play a central role in cellular signal
transduction. As such, alterations in kinase expression and/or
activation can have profound effects on a plethora of cellular
processes, including, but not limited to, activation or inhibition
of cell specific responses, proliferation, and programmed cell
death (apoptosis). Over expression of cloned kinases or of
catalytically inactive mutants of kinases has been used to identify
the role a particular kinase plays in mediating specific signaling
events.
[0120] Kinase mediated cellular signaling often involves a
molecular activation cascade, during which an activated kinase
propagates a ligand-receptor mediated signal by specifically
phosphorylating target substrates. These substrates can themselves
be kinases which become activated following phosphorylation.
Alternatively, they can be adaptor molecules that facilitate down
stream signaling through protein-protein interaction following
phosphorylation. Regardless of the nature of the substrate
molecule(s), expressed catalytically active versions of the
putative kinases in the invention can be used to identify what
substrate(s) were recognized and phosphorylated by the kinase(s) of
the invention. As such, these kinases can be used as reagents to
identify novel molecules involved in signal transduction
pathways.
[0121] Knowledge of a particular kinase would enable one to
enzymatically label the substrate with .sup.32P thereby
facilitating identification and also to use the resultant
radiolabeled protein or a peptide derived from a certain region of
the protein as a substrate probe to identify and isolate specific
phosphatases. Phosphatases are enzymes whose function is to remove
phosphates from selected phosphoproteins; they perform the reverse
function of kinases.
[0122] In some systems specific glycosylations with N-acetyl
glucosamine residues have been described as a biological counter
balance to kinases. That is, glycosidases have been suggested to
compete with kinases for the same serine or threonine residues to
covalently modify. Therefore, the kinase can be used to dissect
dynamic interactions between kinase and phosphatase, and also
between kinase and glycosidase, and finally to examine the dynamic
interactions among kinase, phosphatase and glycosidase
together.
[0123] Kinases phosphorylate target serine, threonine or tyrosine
residues in the context of specific recognition motifs. Recognition
motifs can consist solely of primary structure or in some cases
recognition requires more complex structural features. One can take
advantage of kinases with strict primary sequence recognition
requirements by using them as a general labeling reagent.
Nucleotides coding for the amino acids recognized by a particular
kinase could be engineered onto either end of a protein on
interest, thereby "tagging" the molecule. The expressed, tagged
protein could be .sup.32P-labeled at a known site on the engineered
tag by its specific kinase, thus generating a well defined,
radiolabeled protein.
[0124] Because kinases are phosphotransferases, they must take part
in protein-protein interactions with at least one or more substrate
molecules, i.e. its phosphate recipient(s). Therefore, kinases or
polypeptides comprised of portions of a kinase could be used as
"baits" in the yeast two hybrid system by well established
molecular biology techniques, to identify molecules that interact
directly with the polypeptide.
[0125] Alternatively, polypeptides of the invention could be
engineered prior to expression with a tag such as poly-His or FLAG,
then be expressed and purified using either nickel chelate
chromatography or anti-FLAG antibody coupled to a resin,
respectively. Once bound to the resin, the polypeptide of the
invention could be covalently attached using a bifunctional
cross-linking agent using well established techniques. The
covalently bound polypeptide to the resin could then be used to
purify molecules from cell lysates or cell supernatants (following
treatment with various reagent) through their affinity for the
polypeptide of the invention.
[0126] Isolated and purified kinase polypeptides or a fragment
thereof of the invention can also be useful as a therapeutic agent
in inhibiting signaling. Polypeptides are introduced into the
intracellular environment by well-known means, such as by encasing
the protein in liposomes or coupling it to a monoclonal antibody
targeted to a specific cell type.
[0127] DNA, polypeptides, and antibodies against polypeptides of
the invention can be used as reagents in a variety of research
protocols. A sample of such research protocols are given in
Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol.
1-3, Cold Spring Harbor Laboratory Press, (1989). For example,
these reagents can serve as markers for cell specific or tissue
specific expression of RNA or proteins. Similarly, these reagents
can be used to investigate constitutive and transient expression of
RNA or polypeptides. The DNA can be used to determine the
chromosomal location of DNA and to map genes in relation to this
chromosomal location. The DNA can also be used to examine genetic
heterogeneity and heredity through the use of techniques such as
genetic fingerprinting, as well as to identify risks associated
with genetic disorders. The DNA can be further used to identify
additional genes related to the DNA and to establish evolutionary
trees based on the comparison of sequences. The DNA and
polypeptides can be used to select for those genes or proteins that
are homologous to the DNA or polypeptides, through positive
screening procedures such as Southern blotting and immunoblotting
and through negative screening procedures such as subtraction.
[0128] The polypeptides and fragments of the invention can also be
used as a reagent to identify (a) any protein that polypeptide
regulates, and (b) other proteins with which it might interact.
Polypeptides could be used by coupling recombinant protein to an
affinity matrix, or by using them as a bait in the 2-hybrid system.
The polypeptides and fragments thereof can be used as reagents in
the study of the kinase signaling pathway as a reagent to block
kinase signaling.
[0129] A hallmark of protein kinases is their ability to
phosphorylate other proteins and to auto-phosphorylate. Therefore,
in one aspect of the invention, the isolated polypeptides with
kinase activity can be used in assays to phosphorylate target
proteins, radiolabel target proteins with .sup.32P, and identify
proteins having phosphatase activity. Exemplary methods of
phosphorylation assays set forth above are disclosed in U.S. Pat.
No. 5,447,860 which is incorporated herein by reference. In
addition to full length polypeptides, the invention also includes
the isolated active kinase domains of kinases, such as the
intracellular domain of transmembrane bound kinases and the
cytoplasmic kinases, which can function as reagents in kinase
assays. Further, soluble forms of the extracellular domains of the
kinases are useful in inhibiting the natural ligand-receptor
interaction.
[0130] When used as a therapeutic agent, polypeptides of the
invention can be formulated into pharmaceutical compositions
according to known methods. The polypeptides can be combined in
admixture, either as the sole active material or with other known
active materials, with pharmaceutically suitable diluents (e.g.,
Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal,
benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants
and/or carriers. Suitable carriers and their formulations are
described in Remington's Pharmaceutical Sciences, 16th ed. 1980,
Mack Publishing Co. In addition, such compositions can contain the
polypeptides complexed with polyethylene glycol (PEG), metal ions,
or incorporated into polymeric compounds such as polyacetic acid,
polyglycolic acid, hydrogels, etc., or incorporated into liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or spheroblasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance of polypeptides of the
invention.
[0131] The dosage of the composition can be readily determined by
those of ordinary skill in the art. The amount to be administered
and the frequency of administration can be determined empirically
and will take into consideration the age and size of the patient
being treated, as well as the malady being treated.
[0132] Treatment comprises administering the composition by any
method familiar to those of ordinary skill in the art, including
intravenous, intraperitoneal, intracorporeal injection,
intra-articular, intraventricular, intrathecal, intramuscular,
subcutaneous, topically, tonsillar, intranasally, intravaginally,
and orally. The composition may also be given locally, such as by
injection into the particular area, either intramuscularly or
subcutaneously.
[0133] Within the therapeutic and research aspects of the
invention, polypeptides of the invention, and peptides based on the
amino acid sequence thereof, can be utilized to prepare antibodies
that specifically bind to the polypeptides. The term "antibodies"
is meant to include polyclonal antibodies, monoclonal antibodies,
fragments thereof such as F(ab')2, and Fab fragments, as well as
any recombinantly produced binding partners. Antibodies are defined
to be specifically binding if they bind polypeptides of the
invention with a K.sub.a of greater than or equal to about 10.sup.7
M.sup.-1. Affinities of binding partners or antibodies can be
readily determined using conventional techniques, for example those
described by Scatchard et al., Ann. N. YAcad. Sci., 51:660
(1949).
[0134] Polyclonal antibodies can be readily generated from a
variety of sources, for example, horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, or rats, using procedures that are
well-known in the art. In general, purified polypeptides of the
invention, or a peptide based on the amino acid sequence of
polypeptides of the invention that is appropriately conjugated, is
administered to the host animal typically through parenteral
injection. The immunogenicity of these polypeptides can be enhanced
through the use of an adjuvant, for example, Freund's complete or
incomplete adjuvant. Following booster immunizations, small samples
of serum are collected and tested for reactivity to the
polypeptides. Examples of various assays useful for such
determination include those described in: Antibodies: A Laboratory
Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory
Press, 1988; as well as procedures such as countercurrent
immuno-electrophoresis (CIEP), radioimmunoassay,
radio-immunoprecipitation, enzyme-linked immuno-sorbent assays
(ELISA), dot blot assays, and sandwich assays, see U.S. Pat. Nos.
4,376,110 and 4,486,530.
[0135] Monoclonal antibodies can be readily prepared using
well-known procedures, see for example, the procedures described in
U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993;
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.
Briefly, the host animals, such as Balb/c mice are injected
intraperitoneally at least once, and preferably at least twice at
about 3 week intervals with isolated and purified polypeptides or
conjugated polypeptides of the invention, optionally in the
presence of adjuvant. 10 .mu.g of isolated and purified polypeptide
of the invention or peptides based on the amino acid sequence of
polypeptides of the invention in the presence of RIBI adjuvant
(RIBI Corp., Hamilton, Mont.). Mouse sera are then assayed by
conventional dot blot technique or antibody capture (ABC) to
determine which animal produces the highest level of antibody and
whose spleen cells are the best candidate for fusion. Approximately
two to three weeks later, the mice are given an intravenous boost
of the polypeptides or conjugated polypeptides such as 3 .mu.g
suspended in sterile PBS. Mice are later sacrificed and spleen
cells fused with commercially available myeloma cells, such as
Ag8.653 (ATCC), following established protocols. Briefly, the
myeloma cells are washed several times in media and fused to mouse
spleen cells at a ratio of about three spleen cells to one myeloma
cell. The fusing agent can be any suitable agent used in the art,
for example, polyethylene glycol (PEG) or more preferably, 50% PEG:
10% DMSO (Sigma). Fusion is plated out into, for example, twenty
96-well flat bottom plates (Corning) containing an appropriate
medium, such as HAT supplemented DMEM media and allowed to grow for
eight days. Supernatants from resultant hybridomas are collected
and added to, for example, a 96-well plate for 60 minutes that is
first coated with goat anti-mouse Ig. Following washes,
.sup.125I-polypeptide or peptides of the invention are added to
each well, incubated for 60 minutes at room temperature, and washed
four times. Positive wells can be subsequently detected by
conventional methods, such as autoradiography at -70.degree. C.
using Kodak X-Omat S film. Positive clones can be grown in bulk
culture and supernatants are subsequently purified, such as over a
Protein A column (Pharmacia). It is understood of course that many
techniques could be used to generate antibodies against
polypeptides and fragmented peptides of the invention and that this
embodiment in no way limits the scope of the invention.
[0136] The monoclonal antibodies of the invention can be produced
using alternative techniques, such as those described by
Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A
Rapid Alternative to Hybridomas", Strategies in Molecular Biology
3:1-9 (1990), which is incorporated herein by reference. Similarly,
binding partners can be constructed using recombinant DNA
techniques to incorporate the variable regions of a gene that
encodes a specific binding antibody. Such a technique is described
in Larrick et al., Biotechnology, 7:394 (1989).
[0137] Other types of "antibodies" can be produced using the
information provided herein in conjunction with the state of
knowledge in the art. For example, antibodies that have been
engineered to contain elements of human antibodies that are capable
of specifically binding polypeptides of the invention are also
encompassed by the invention.
[0138] Once isolated and purified, the antibodies against
polypeptides of the invention can be used to detect the presence of
the polypeptides in a sample using established assay protocols.
Further, the antibodies of the invention can be used
therapeutically or for research purposes to bind to the
polypeptides and inhibit its activity in vivo or in vitro.
[0139] Antibodies immunoreactive with polypeptides of the
invention, and in particular, monoclonal antibodies against these
polypeptides, are now made available through the invention. Such
antibodies can be useful for inhibiting polypeptide activity in
vivo and for detecting the presence of polypeptides of the
invention in a sample.
[0140] In another embodiment, antibodies generated against a
polypeptide and fragmented peptides of the invention can be used in
combination with polypeptide or fragmented peptide molecular weight
markers of the invention to enhance the accuracy in the use of
these molecular weight markers to determine the apparent molecular
weight and isoelectric point of a sample protein. Polypeptide or
fragmented peptide molecular weight markers of the invention can be
mixed with a molar excess of a sample protein and the mixture can
be resolved by two dimensional electrophoresis by conventional
means. Polypeptides can be transferred to a suitable protein
binding membrane, such as nitrocellulose, by conventional means and
detected by the antibodies of the invention.
[0141] The purified polypeptides according to the invention will
facilitate the discovery of inhibitors of such polypeptides. The
use of a purified polypeptide of the invention in the screening of
potential inhibitors thereof is important and can eliminate or
reduce the possibility of interfering reactions with
contaminants.
[0142] In addition, polypeptides of the invention can be used for
structure-based design of polypeptide-inhibitors. Such
structure-based design is also known as "rational drug design." The
polypeptides can be three-dimensionally analyzed by, for example,
X-ray crystallography, nuclear magnetic resonance or homology
modeling, all of which are well-known methods. The use of the
polypeptide structural information in molecular modeling software
systems to assist in inhibitor design and inhibitor-polypeptide
interaction is also encompassed by the invention. Such
computer-assisted modeling and drug design can utilize information
such as chemical conformational analysis, electrostatic potential
of the molecules, protein folding, etc. For example, most of the
design of class-specific inhibitors of metalloproteases has focused
on attempts to chelate or bind the catalytic zinc atom. Synthetic
inhibitors are usually designed to contain a negatively-charged
moiety to which is attached a series of other groups designed to
fit the specificity pockets of the particular protease. A
particular method of the invention comprises analyzing the three
dimensional structure of polypeptides of the invention for likely
binding sites of substrates, synthesizing a new molecule that
incorporates a predictive reactive site, and assaying the new
molecule as described above.
[0143] The polypeptides of the present invention may also be used
in a screening assay to identify compounds and small molecules
which inhibit (antagonize) or enhance (agonize) activation of the
polypeptides of the instant invention. Thus, for example,
polypeptides of the invention may be used to identify antagonists
and agonists from cells, cell-free preparations, chemical
libraries, and natural product mixtures. The antagonists and
agonists may be natural or modified substrates, ligands, enzymes,
receptors, etc. of the polypeptides of the instant invention, or
may be structural or functional mimetics of the polypeptides.
Potential antagonists of the polypeptides of the instant invention
may include small molecules, peptides, and antibodies that bind to
and occupy a binding site of the polypeptides, causing them to be
unavailable to bind to their ligands and therefore preventing
normal biological activity. Other potential antagonists are
antisense molecules which may hybridize to mRNA in vivo and block
translation of the mRNA into the polypeptides of the instant
invention. Potential agonists include small molecules, peptides and
antibodies which bind to the instant polypeptides and elicit the
same or enhanced biological effects as those caused by the binding
of the polypeptides of the instant invention.
[0144] Small molecule agonists and antagonists are usually less
than 10K molecular weight and may possess a number of
physiochemical and pharmacological properties that enhance cell
penetration, resist degradation and prolong their physiological
half-lives. (Gibbs, J., Pharmaceutical Research in Molecular
Oncology, Cell, Vol. 79 (1994).) Antibodies, which include intact
molecules as well as fragments such as Fab and F(ab')2 fragments,
may be used to bind to and inhibit the polypeptides of the instant
invention by blocking the commencement of a signaling cascade. It
is preferable that the antibodies are humanized, and more
preferable that the antibodies are human. The antibodies of the
present invention may be prepared by any of a variety of well-known
methods.
[0145] Specific screening methods are known in the art and many are
extensively incorporated in high throughput test systems so that
large numbers of test compounds can be screened within a short
amount of time. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays, cell based assays, etc. These assay
formats are well known in the art. The screening assays of the
present invention are amenable to screening of chemical libraries
and are suitable for the identification of small molecule drug
candidates, antibodies, peptides and other antagonists and
agonists.
[0146] One embodiment of a method for identifying molecules which
antagonize or inhibit the polypeptides involves adding a candidate
molecule to a medium which contains cells that express the
polypeptides of the instant invention; changing the conditions of
said medium so that, but for the presence of the candidate
molecule, the polypeptides would be bound to their ligands; and
observing the binding and stimulation or inhibition of a functional
response. The activity of the cells which were contacted with the
candidate molecule may then be compared with the identical cells
which were not contacted and agonists and antagonists of the
polypeptides of the instant invention may be identified. The
measurement of biological activity may be performed by a number of
well-known methods such as measuring the amount of protein present
(e.g. an ELISA) or of the protein's activity. A decrease in
biological stimulation or activation would indicate an antagonist.
An increase would indicate an agonist. Specifically, one embodiment
of the instant invention includes agonists and antagonists of
QQ3351, SS1771, SS1771A and truncated QQ3351.
[0147] Screening assays can further be designed to find molecules
that mimic the biological activity of the polypeptides of the
instant invention. Molecules which mimic the biological activity of
a polypeptide may be useful for enhancing the biological activity
of the polypeptide. To identify compounds for therapeutically
active agents that mimic the biological activity of a polypeptide,
it must first be determined whether a candidate molecule binds to
the polypeptide. A binding candidate molecule is then added to a
biological assay to determine its biological effects. The
biological effects of the candidate molecule are then compared to
the those of the polypeptide.
[0148] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification,
which are hereby incorporated by reference. The embodiments within
the specification provide an illustration of embodiments of the
invention and should not be construed to limit the scope of the
invention. The skilled artisan recognizes many other embodiments
are encompassed by the claimed invention.
Sequence CWU 1
1
16 1 181 DNA Homo sapiens 1 gtacgccatg aaggtgctgc gcaaggcggc
gctggtgcag cgcgccaaga cgcaagagca 60 cacgcgcacc gagcgctcgg
tgctggagct ggtgcgccag gcgcccttcc tggtcacgct 120 gcactacgct
ttccagacgg atgccaagct gcacctcatc ctggactatg tgagcggcgg 180 g 181 2
221 DNA Homo sapiens 2 cccgagaggt gccacatcag accgcctccg acttcgtgcg
ggactcggcg gccagccacc 60 aggcggagcc cgaggcgtac gagcggcgcg
tgtgcttcct gcttctgcaa ctctgcaacg 120 ggctggagca cctgaaggag
cacgggatca tccaccggga cctgtgcctg gagaacctgc 180 tgctggtgca
ctgcaccctc caggccggcc ccgggcccgc c 221 3 1085 DNA Homo sapiens 3
cgggcagggc tggagctggg ctgggatccc gagctcggca gcagcgcagc gggccggccc
60 acctgctggt gccctggagg ctctgagccc cggcggcgcc cgggcccacg
cggaacgacg 120 gggcgagatg cgagccaccc ctctggctgc tcctgcgggt
tccctgtcca ggaagaagcg 180 gttggagttg gatgacaact tagataccga
gcgtcccgtc cagaaacgag ctcgaagtgg 240 gccccagccc agactgcccc
cctgcctgtt gcccctgagc ccacctactg ctccagatcg 300 tgcaactgct
gtggccactg cctcccgtct tgggccctat gtcctcctgg agcccgagga 360
gggcgggcgg gcctaccagg ccctgcactg ccctacaggc actgagtata cctgcaaggt
420 gtaccccgtc caggaagccc tggccgtgct ggagccctac gcgcggctgc
ccccgcacaa 480 gcatgtggct cggcccactg aggtcctggc tggtacccag
ctcctctacg cctttttcac 540 tcggacccat ggggacatgc acagcctggt
gcgaagccgc caccgtatcc ctgagcctga 600 ggctgccgtg ctcttccgcc
agatggccac cgccctggcg cactgtcacc agcacggtct 660 ggtcctgcgt
gatctcaagc tgtgtcgctt tgtcttcgct gaccgtgaga ggaagaagct 720
ggtgctggag aacctggagg actcctgcgt gctgactggg ccagatgatt ccctgtggga
780 caagcacgcg tgcccagcct acgtgggacc tgagatactc agctcacggg
cctcatactc 840 gggcaaggca gccgatgtct ggagcctggg cgtggcgctc
ttcaccatgc tggccggcca 900 ctaccccttc caggactcgg agcctgtcct
gctcttcggc aagatccgcc gcggggccta 960 cgccttgcct gcaggcctct
cggcccctgc ccgctgtctg gttcgctgcc tccttcgtcg 1020 ggagccagct
gaacggctca cagccacagg catcctcctg cacccctggc tgcgacagga 1080 cccga
1085 4 388 DNA Homo sapiens 4 cagcgagaag ccgacatgca tcgcctcttc
aatcacccca acatccttcg cctcgtggct 60 tactgtctga gggaacgggg
tgctaagcat gaggcctggc tgctgctacc attcttcaag 120 agaggtacgc
tgtggaatga gatagaaagg ctgaaggaca aaggcaactt cctgaccgag 180
gatcaaatcc tttggctgct gctggggatc tgcagaggcc ttgaggccat tcatgccaag
240 ggttatgcct acagagactt gaagcccacc aatatattgc ttggagatga
ggggcagcca 300 gttttaatgg acttgggttc catgaatcaa gcatgcatcc
atgtggaggg ctcccgccag 360 gctctgaccc tgcaggactg ggcagccc 388 5 1555
DNA Homo sapiens 5 atgctaacta gtttaaacag atcttggaac gagacgacct
gctgtggaag agcgagcttt 60 ttggaactgt gcacgggaca gattggacgc
acacccctcg ggaggcgcga aggcatggaa 120 aatttgaagc atattatcac
ccttggccag gtcatccaca aacggtgtga agagatgaaa 180 tactgcaaga
aacagtgccg gcgcctgggc caccgcgtcc tcggcctgat caagcctctg 240
gagatgctcc aggaccaagg aaagaggagc gtgccctctg agaagttaac cacagccatg
300 aaccgcttca aggctgccct ggaggaggct aatggggaga tagaaaagtt
cagcaataga 360 tccaatatct gcaggtttct aacagcaagc caggacaaaa
tactcttcaa ggacgtgaac 420 aggaagctga gtgatgtctg gaaggagctc
tcgctgttac ttcaggttga gcaacgcatg 480 cctgtttcac ccataagcca
aggagcgtcc tgggcacagg aagatcagca ggatgcagac 540 gaagacaggc
gagctttcca gatgctaaga agagataatg aaaaaataga agcttcactg 600
agacgattag aaatcaacat gaaagaaatc aaggaaactt tgaggcagta tttaccacca
660 aaatgcatgc aggagatccc gcaagagcaa atcaaggaga tcaagaagga
gcagctttca 720 ggatccccgt ggattctgct aagggaaaat gaagtcagca
cactttataa aggagaatac 780 cacagagctc cagtggccat aaaagtattc
aaaaaactcc aggctggcag cattgcaata 840 gtgaggcaga ctttcaataa
ggagatcaaa accatgaaga aattcgaatc tcccaacatc 900 ctgcgtatat
ttgggatttg cattgatgaa acagtgactc cgcctcaatt ctccattgtc 960
atggagtact gtgaactcgg gaccctgagg gagctgttgg atagggaaaa agacctcaca
1020 cttggcaagc gcatggtcct agtcctgggg gcagcccgag gcctataccg
gctacaccat 1080 tcagaagcac ctgaactcca cggaaaaatc agaagctcaa
acttcctggt aactcaaggc 1140 taccaagtga agcttgcagg atttgagttg
aggaaaacac agacttccat gagtttggga 1200 actacgagag aaaagacaga
cagagtcaaa tctacagcat atctctcacc tcaggaactg 1260 gaagatgtat
tttatcaata tgatgtaaag tctgaaatat acagctttgg aatcgtcctc 1320
tgggaaatcg ccactggaga tatcccgttt caaggctgta attctgagaa gatccgcaag
1380 ctggtggctg tgaagcggca gcaggagcca ctgggtgaag actgcccttc
agagctgcgg 1440 gagatcattg atgagtgccg ggcagcaggt cgtctcgttc
caagatctgt agcggccgcc 1500 cgggccgtcg acgtttaaac gcgtggccct
cgagaggttt tccgatccgg tcgat 1555 6 1498 DNA Homo sapiens 6
cttcccgctg gacgtggagt acggaggccc agaccggagg tgcccgcctc cgccctaccc
60 gaagcacctg ctgctgcgca gcaagtcgga gcagtacgac ctggacagcc
tgtgcgcagg 120 catggagcag agcctccgtg cgggccccaa cgagcccgag
ggcggcgaca agagccgcaa 180 aagcgccaag ggggacaaag gcggaaagga
taaaaagcag attcagacct ctcccgttcc 240 cgtccgcaaa aacagcagag
acgaagagaa gagagagtca cgcatcaaga gctactcgcc 300 atacgccttt
aagttcttca tggagcagca cgtggagaat gtcatcaaaa cctaccagca 360
gaaggttaac cggaggctgc agctggagca agaaatggcc aaagctggac tctgtgaagc
420 tgagcaggag cagatgcgga agatcctcta ccagaaagag tctaattaca
acaggttaaa 480 gagggccaag atggacaagt ctatgtttgt caagatcaaa
accctgggga tcggtgcctt 540 tggagaagtg tgccttgctt gtaaggtgga
cactcacgcc ctgtacgcca tgaagaccct 600 aaggaaaaag gatgtcctga
accggaatca ggtggcccac gtcaaggccg agagggacat 660 cctggccgag
gcagacaatg agtgggtggt caaactctac tactccttcc aagacaaaga 720
cagcctgtac tttgtgatgg actacatccc tggtggggac atgatgagcc tgctgatccg
780 gatggaggtc ttccctgagc acctggcccg gttctacatc gcagagctga
ctttggccat 840 tgagagtgtc cacaagatgg gcttcatcca ccgagacatc
aagcctgata acattttgat 900 agatctggat ggtcacatta aactcacaga
tttcggcctc tgcactgggt tcaggtggac 960 tcacaattcc aaatattacc
agaaagggag ccatgtcaga caggacagca tggagcccag 1020 cgacctctgg
gatgatgtgt ctaactgtcg gtgtggggac aggctgaaga ccctagagca 1080
gagggcgcgg aagcagcacc agaggtgcct ggcacattca ctggtgggga ctccaaacta
1140 catcgcaccc gaggtgctcc tccgcaaagg gtacactcaa ctctgtgact
ggtggagtgt 1200 tggagtgatt ctcttcgaga tgctggtggg gcagccgccc
tttttggcac ctactcccac 1260 agaaacccag ctgaaggtga tcaactggga
gaacacgctc cacattccag cccaggtgaa 1320 gctgagccct gaggccaggg
acctcatcac caagctgtgc tgctccgcag accaccgcct 1380 ggggcggaat
ggggccgatg acctgaaggc ccaccccttc ttcagcgcca ttgacttctc 1440
cagtgacatc cggaagcatc cagcccccta cgttcccacc atcagccacc ccatggag
1498 7 60 PRT Homo sapiens 7 Tyr Ala Met Lys Val Leu Arg Lys Ala
Ala Leu Val Gln Arg Ala Lys 1 5 10 15 Thr Gln Glu His Thr Arg Thr
Glu Arg Ser Val Leu Glu Leu Val Arg 20 25 30 Gln Ala Pro Phe Leu
Val Thr Leu His Tyr Ala Phe Gln Thr Asp Ala 35 40 45 Lys Leu His
Leu Ile Leu Asp Tyr Val Ser Gly Gly 50 55 60 8 73 PRT Homo sapiens
8 Arg Glu Val Pro His Gln Thr Ala Ser Asp Phe Val Arg Asp Ser Ala 1
5 10 15 Ala Ser His Gln Ala Glu Pro Glu Ala Tyr Glu Arg Arg Val Cys
Phe 20 25 30 Leu Leu Leu Gln Leu Cys Asn Gly Leu Glu His Leu Lys
Glu His Gly 35 40 45 Ile Ile His Arg Asp Leu Cys Leu Glu Asn Leu
Leu Leu Val His Cys 50 55 60 Thr Leu Gln Ala Gly Pro Gly Pro Ala 65
70 9 360 PRT Homo sapiens 9 Gly Gln Gly Trp Ser Trp Ala Gly Ile Pro
Ser Ser Ala Ala Ala Gln 1 5 10 15 Arg Ala Gly Pro Pro Ala Gly Ala
Leu Glu Ala Leu Ser Pro Gly Gly 20 25 30 Ala Arg Ala His Ala Glu
Arg Arg Gly Glu Met Arg Ala Thr Pro Leu 35 40 45 Ala Ala Pro Ala
Gly Ser Leu Ser Arg Lys Lys Arg Leu Glu Leu Asp 50 55 60 Asp Asn
Leu Asp Thr Glu Arg Pro Val Gln Lys Arg Ala Arg Ser Gly 65 70 75 80
Pro Gln Pro Arg Leu Pro Pro Cys Leu Leu Pro Leu Ser Pro Pro Thr 85
90 95 Ala Pro Asp Arg Ala Thr Ala Val Ala Thr Ala Ser Arg Leu Gly
Pro 100 105 110 Tyr Val Leu Leu Glu Pro Glu Glu Gly Gly Arg Ala Tyr
Gln Ala Leu 115 120 125 His Cys Pro Thr Gly Thr Glu Tyr Thr Cys Lys
Val Tyr Pro Val Gln 130 135 140 Glu Ala Leu Ala Val Leu Glu Pro Tyr
Ala Arg Leu Pro Pro His Lys 145 150 155 160 His Val Ala Arg Pro Thr
Glu Val Leu Ala Gly Thr Gln Leu Leu Tyr 165 170 175 Ala Phe Phe Thr
Arg Thr His Gly Asp Met His Ser Leu Val Arg Ser 180 185 190 Arg His
Arg Ile Pro Glu Pro Glu Ala Ala Val Leu Phe Arg Gln Met 195 200 205
Ala Thr Ala Leu Ala His Cys His Gln His Gly Leu Val Leu Arg Asp 210
215 220 Leu Lys Leu Cys Arg Phe Val Phe Ala Asp Arg Glu Arg Lys Lys
Leu 225 230 235 240 Val Leu Glu Asn Leu Glu Asp Ser Cys Val Leu Thr
Gly Pro Asp Asp 245 250 255 Ser Leu Trp Asp Lys His Ala Cys Pro Ala
Tyr Val Gly Pro Glu Ile 260 265 270 Leu Ser Ser Arg Ala Ser Tyr Ser
Gly Lys Ala Ala Asp Val Trp Ser 275 280 285 Leu Gly Val Ala Leu Phe
Thr Met Leu Ala Gly His Tyr Pro Phe Gln 290 295 300 Asp Ser Glu Pro
Val Leu Leu Phe Gly Lys Ile Arg Arg Gly Ala Tyr 305 310 315 320 Ala
Leu Pro Ala Gly Leu Ser Ala Pro Ala Arg Cys Leu Val Arg Cys 325 330
335 Leu Leu Arg Arg Glu Pro Ala Glu Arg Leu Thr Ala Thr Gly Ile Leu
340 345 350 Leu His Pro Trp Leu Arg Gln Asp 355 360 10 146 PRT Homo
sapiens UNSURE (140)..(140) UNSURE 10 Gln Arg Glu Ala Asp Met His
Arg Leu Phe Asn His Pro Asn Ile Leu 1 5 10 15 Arg Leu Val Ala Tyr
Cys Leu Arg Glu Arg Gly Ala Lys His Glu Ala 20 25 30 Trp Leu Leu
Leu Pro Phe Phe Lys Arg Gly Thr Leu Trp Asn Glu Ile 35 40 45 Glu
Arg Leu Lys Asp Lys Gly Asn Phe Leu Thr Glu Asp Gln Ile Leu 50 55
60 Trp Leu Leu Leu Gly Ile Cys Arg Gly Leu Glu Ala Ile His Ala Lys
65 70 75 80 Gly Tyr Ala Tyr Arg Asp Leu Lys Pro Thr Asn Ile Leu Leu
Gly Asp 85 90 95 Glu Gly Gln Pro Val Leu Met Asp Leu Gly Ser Met
Asn Gln Ala Cys 100 105 110 Ile His Val Glu Gly Ser Arg Gln Ala Leu
Thr Leu Gln Asp Trp Ala 115 120 125 Ala Gln Arg Cys Thr Ile Ser Tyr
Arg Ala Pro Xaa Leu Phe Ser Val 130 135 140 Gln Ser 145 11 505 PRT
Homo sapiens 11 Met Leu Thr Ser Leu Asn Arg Ser Trp Asn Glu Thr Thr
Cys Cys Gly 1 5 10 15 Arg Ala Ser Phe Leu Glu Leu Cys Thr Gly Gln
Ile Gly Arg Thr Pro 20 25 30 Leu Gly Arg Arg Glu Gly Met Glu Asn
Leu Lys His Ile Ile Thr Leu 35 40 45 Gly Gln Val Ile His Lys Arg
Cys Glu Glu Met Lys Tyr Cys Lys Lys 50 55 60 Gln Cys Arg Arg Leu
Gly His Arg Val Leu Gly Leu Ile Lys Pro Leu 65 70 75 80 Glu Met Leu
Gln Asp Gln Gly Lys Arg Ser Val Pro Ser Glu Lys Leu 85 90 95 Thr
Thr Ala Met Asn Arg Phe Lys Ala Ala Leu Glu Glu Ala Asn Gly 100 105
110 Glu Ile Glu Lys Phe Ser Asn Arg Ser Asn Ile Cys Arg Phe Leu Thr
115 120 125 Ala Ser Gln Asp Lys Ile Leu Phe Lys Asp Val Asn Arg Lys
Leu Ser 130 135 140 Asp Val Trp Lys Glu Leu Ser Leu Leu Leu Gln Val
Glu Gln Arg Met 145 150 155 160 Pro Val Ser Pro Ile Ser Gln Gly Ala
Ser Trp Ala Gln Glu Asp Gln 165 170 175 Gln Asp Ala Asp Glu Asp Arg
Arg Ala Phe Gln Met Leu Arg Arg Asp 180 185 190 Asn Glu Lys Ile Glu
Ala Ser Leu Arg Arg Leu Glu Ile Asn Met Lys 195 200 205 Glu Ile Lys
Glu Thr Leu Arg Gln Tyr Leu Pro Pro Lys Cys Met Gln 210 215 220 Glu
Ile Pro Gln Glu Gln Ile Lys Glu Ile Lys Lys Glu Gln Leu Ser 225 230
235 240 Gly Ser Pro Trp Ile Leu Leu Arg Glu Asn Glu Val Ser Thr Leu
Tyr 245 250 255 Lys Gly Glu Tyr His Arg Ala Pro Val Ala Ile Lys Val
Phe Lys Lys 260 265 270 Leu Gln Ala Gly Ser Ile Ala Ile Val Arg Gln
Thr Phe Asn Lys Glu 275 280 285 Ile Lys Thr Met Lys Lys Phe Glu Ser
Pro Asn Ile Leu Arg Ile Phe 290 295 300 Gly Ile Cys Ile Asp Glu Thr
Val Thr Pro Pro Gln Phe Ser Ile Val 305 310 315 320 Met Glu Tyr Cys
Glu Leu Gly Thr Leu Arg Glu Leu Leu Asp Arg Glu 325 330 335 Lys Asp
Leu Thr Leu Gly Lys Arg Met Val Leu Val Leu Gly Ala Ala 340 345 350
Arg Gly Leu Tyr Arg Leu His His Ser Glu Ala Pro Glu Leu His Gly 355
360 365 Lys Ile Arg Ser Ser Asn Phe Leu Val Thr Gln Gly Tyr Gln Val
Lys 370 375 380 Leu Ala Gly Phe Glu Leu Arg Lys Thr Gln Thr Ser Met
Ser Leu Gly 385 390 395 400 Thr Thr Arg Glu Lys Thr Asp Arg Val Lys
Ser Thr Ala Tyr Leu Ser 405 410 415 Pro Gln Glu Leu Glu Asp Val Phe
Tyr Gln Tyr Asp Val Lys Ser Glu 420 425 430 Ile Tyr Ser Phe Gly Ile
Val Leu Trp Glu Ile Ala Thr Gly Asp Ile 435 440 445 Pro Phe Gln Gly
Cys Asn Ser Glu Lys Ile Arg Lys Leu Val Ala Val 450 455 460 Lys Arg
Gln Gln Glu Pro Leu Gly Glu Asp Cys Pro Ser Glu Leu Arg 465 470 475
480 Glu Ile Ile Asp Glu Cys Arg Ala Ala Gly Arg Leu Val Pro Arg Ser
485 490 495 Val Ala Ala Ala Arg Ala Val Asp Val 500 505 12 499 PRT
Homo sapiens 12 Phe Pro Leu Asp Val Glu Tyr Gly Gly Pro Asp Arg Arg
Cys Pro Pro 1 5 10 15 Pro Pro Tyr Pro Lys His Leu Leu Leu Arg Ser
Lys Ser Glu Gln Tyr 20 25 30 Asp Leu Asp Ser Leu Cys Ala Gly Met
Glu Gln Ser Leu Arg Ala Gly 35 40 45 Pro Asn Glu Pro Glu Gly Gly
Asp Lys Ser Arg Lys Ser Ala Lys Gly 50 55 60 Asp Lys Gly Gly Lys
Asp Lys Lys Gln Ile Gln Thr Ser Pro Val Pro 65 70 75 80 Val Arg Lys
Asn Ser Arg Asp Glu Glu Lys Arg Glu Ser Arg Ile Lys 85 90 95 Ser
Tyr Ser Pro Tyr Ala Phe Lys Phe Phe Met Glu Gln His Val Glu 100 105
110 Asn Val Ile Lys Thr Tyr Gln Gln Lys Val Asn Arg Arg Leu Gln Leu
115 120 125 Glu Gln Glu Met Ala Lys Ala Gly Leu Cys Glu Ala Glu Gln
Glu Gln 130 135 140 Met Arg Lys Ile Leu Tyr Gln Lys Glu Ser Asn Tyr
Asn Arg Leu Lys 145 150 155 160 Arg Ala Lys Met Asp Lys Ser Met Phe
Val Lys Ile Lys Thr Leu Gly 165 170 175 Ile Gly Ala Phe Gly Glu Val
Cys Leu Ala Cys Lys Val Asp Thr His 180 185 190 Ala Leu Tyr Ala Met
Lys Thr Leu Arg Lys Lys Asp Val Leu Asn Arg 195 200 205 Asn Gln Val
Ala His Val Lys Ala Glu Arg Asp Ile Leu Ala Glu Ala 210 215 220 Asp
Asn Glu Trp Val Val Lys Leu Tyr Tyr Ser Phe Gln Asp Lys Asp 225 230
235 240 Ser Leu Tyr Phe Val Met Asp Tyr Ile Pro Gly Gly Asp Met Met
Ser 245 250 255 Leu Leu Ile Arg Met Glu Val Phe Pro Glu His Leu Ala
Arg Phe Tyr 260 265 270 Ile Ala Glu Leu Thr Leu Ala Ile Glu Ser Val
His Lys Met Gly Phe 275 280 285 Ile His Arg Asp Ile Lys Pro Asp Asn
Ile Leu Ile Asp Leu Asp Gly 290 295 300 His Ile Lys Leu Thr Asp Phe
Gly Leu Cys Thr Gly Phe Arg Trp Thr 305 310 315 320 His Asn Ser Lys
Tyr Tyr Gln Lys Gly Ser His Val Arg Gln Asp Ser 325 330 335 Met Glu
Pro Ser Asp Leu Trp Asp Asp Val Ser Asn Cys Arg Cys Gly 340 345 350
Asp Arg Leu Lys Thr Leu Glu Gln Arg Ala Arg Lys Gln His Gln Arg 355
360 365 Cys Leu Ala His Ser Leu Val Gly Thr Pro Asn Tyr Ile Ala Pro
Glu 370 375 380 Val Leu Leu Arg Lys Gly Tyr Thr Gln Leu Cys Asp Trp
Trp Ser Val 385 390 395 400 Gly Val Ile Leu Phe Glu Met Leu Val Gly
Gln Pro Pro Phe Leu Ala 405 410 415 Pro Thr Pro Thr Glu Thr Gln Leu
Lys Val Ile Asn Trp Glu Asn Thr
420 425 430 Leu His Ile Pro Ala Gln Val Lys Leu Ser Pro Glu Ala Arg
Asp Leu 435 440 445 Ile Thr Lys Leu Cys Cys Ser Ala Asp His Arg Leu
Gly Arg Asn Gly 450 455 460 Ala Asp Asp Leu Lys Ala His Pro Phe Phe
Ser Ala Ile Asp Phe Ser 465 470 475 480 Ser Asp Ile Arg Lys His Pro
Ala Pro Tyr Val Pro Thr Ile Ser His 485 490 495 Pro Met Glu 13 375
DNA Homo sapiens 13 cttgcaggat ttgagttgag gaaaacacag acttccatga
gtttgggaac tacgagagaa 60 aagacagaca gagtcaaatc tacagcatat
ctctcacctc aggaactgga agatgtattt 120 tatcaatatg atgtaaagtc
tgaaatatac agctttggaa tcgtcctctg ggaaatcgcc 180 actggagata
tcccgtttca aggctgtaat tctgagaaga tccgcaagct ggtggctgtg 240
aagcggcagc aggagccact gggtgaagac tgcccttcag agctgcggga gatcattgat
300 gagtgccggg cccatgatcc ctctgtgcgg ccctctgtgg atgaaatctt
aaagaaactc 360 tccacctttt ctaag 375 14 125 PRT Homo sapiens 14 Leu
Ala Gly Phe Glu Leu Arg Lys Thr Gln Thr Ser Met Ser Leu Gly 1 5 10
15 Thr Thr Arg Glu Lys Thr Asp Arg Val Lys Ser Thr Ala Tyr Leu Ser
20 25 30 Pro Gln Glu Leu Glu Asp Val Phe Tyr Gln Tyr Asp Val Lys
Ser Glu 35 40 45 Ile Tyr Ser Phe Gly Ile Val Leu Trp Glu Ile Ala
Thr Gly Asp Ile 50 55 60 Pro Phe Gln Gly Cys Asn Ser Glu Lys Ile
Arg Lys Leu Val Ala Val 65 70 75 80 Lys Arg Gln Gln Glu Pro Leu Gly
Glu Asp Cys Pro Ser Glu Leu Arg 85 90 95 Glu Ile Ile Asp Glu Cys
Arg Ala His Asp Pro Ser Val Arg Pro Ser 100 105 110 Val Asp Glu Ile
Leu Lys Lys Leu Ser Thr Phe Ser Lys 115 120 125 15 1961 DNA Homo
sapiens 15 tcccgctgga cgtggagtac ggaggcccag accggaggtg cccgcctccg
ccctacccga 60 agcacctgct gctgcgcagc aagtcggagc agtacgacct
ggacagcctg tgcgcaggca 120 tggagcagag cctccgtgcg ggccccaacg
agcccgaggg cggcgacaag agccgcaaaa 180 gcgccaaggg ggacaaaggc
ggaaaggata aaaagcagat tcagacctct cccgttcccg 240 tccgcaaaaa
cagcagagac gaagagaaga gagagtcacg catcaagagc tactcgccat 300
acgcctttaa gttcttcatg gagcagcacg tggagaatgt catcaaaacc taccagcaga
360 aggttaaccg gaggctgcag ctggagcaag aaatggccaa agctggactc
tgtgaagctg 420 agcaggagca gatgcggaag atcctctacc agaaagagtc
taattacaac aggttaaaga 480 gggccaagat ggacaagtct atgtttgtca
agatcaaaac cctggggatc ggtgcctttg 540 gagaagtgtg ccttgcttgt
aaggtggaca ctcacgccct gtacgccatg aagaccctaa 600 ggaaaaagga
tgtcctgaac cggaatcagg tggcccacgt caaggccgag agggacatcc 660
tggccgaggc agacaatgag tgggtggtca aactctacta ctccttccaa gacaaagaca
720 gcctgtactt tgtgatggac tacatccctg gtggggacat gatgagcctg
ctgatccgga 780 tggaggtctt ccctgagcac ctggcccggt tctacatcgc
agagctgact ttggccattg 840 agagtgtcca caagatgggc ttcatccacc
gagacatcaa gcctgataac attttgatag 900 atctggatgg tcacattaaa
ctcacagatt tcggcctctg cactgggttc aggtggactc 960 acaattccaa
atattaccag aaagggagcc atgtcagaca ggacagcatg gagcccagcg 1020
acctctggga tgatgtgtct aactgtcggt gtggggacag gctgaagacc ctagagcaga
1080 gggcgcggaa gcagcaccag aggtgcctgg cacattcact ggtggggact
ccaaactaca 1140 tcgcacccga ggtgctcctc cgcaaagggt acactcaact
ctgtgactgg tggagtgttg 1200 gagtgattct cttcgagatg ctggtggggc
agccgccctt tttggcacct actcccacag 1260 aaacccagct gaaggtgatc
aactgggaga acacgctcca cattccagcc caggtgaagc 1320 tgagccctga
ggccagggac ctcatcacca agctgtgctg ctccgcagac caccgcctgg 1380
ggcggaatgg ggccgatgac ctgaaggccc accccttctt cagcgccatt gacttctcca
1440 gtgacatccg gaagcatcca gccccctacg ttcccaccat cagccacccc
atggacacct 1500 cgaatttcga ccccgtagat gaagaaagcc cttggaacga
tgccagcgaa ggtagcacca 1560 aggcctggga cacactcacc tcgcccaata
acaagcatcc tgagcacgca ttttacgaat 1620 tcaccttccg aaggttcttt
gatgacaatg gctacccctt tcgatgccca aagccttcag 1680 gagcagaagc
ttcacaggct gagagctcag atttagaaag ctctgatctg gtggatcaga 1740
ctgaaggctg ccagcctgtg tacgtgtaga tgggggccag gcacccccac cactcgctgc
1800 ctcccaggtc agggtcccgg agccggtgcc ctcacaggcc aatagggaag
ccgagggctg 1860 ttttgtttta aattagtccg tcgattactt cacttgaaat
tctgctcttc accaagaaaa 1920 cccaaacagg acacttttga aaacagcggt
gccgcgaatt c 1961 16 588 PRT Homo sapiens 16 Pro Leu Asp Val Glu
Tyr Gly Gly Pro Asp Arg Arg Cys Pro Pro Pro 1 5 10 15 Pro Tyr Pro
Lys His Leu Leu Leu Arg Ser Lys Ser Glu Gln Tyr Asp 20 25 30 Leu
Asp Ser Leu Cys Ala Gly Met Glu Gln Ser Leu Arg Ala Gly Pro 35 40
45 Asn Glu Pro Glu Gly Gly Asp Lys Ser Arg Lys Ser Ala Lys Gly Asp
50 55 60 Lys Gly Gly Lys Asp Lys Lys Gln Ile Gln Thr Ser Pro Val
Pro Val 65 70 75 80 Arg Lys Asn Ser Arg Asp Glu Glu Lys Arg Glu Ser
Arg Ile Lys Ser 85 90 95 Tyr Ser Pro Tyr Ala Phe Lys Phe Phe Met
Glu Gln His Val Glu Asn 100 105 110 Val Ile Lys Thr Tyr Gln Gln Lys
Val Asn Arg Arg Leu Gln Leu Glu 115 120 125 Gln Glu Met Ala Lys Ala
Gly Leu Cys Glu Ala Glu Gln Glu Gln Met 130 135 140 Arg Lys Ile Leu
Tyr Gln Lys Glu Ser Asn Tyr Asn Arg Leu Lys Arg 145 150 155 160 Ala
Lys Met Asp Lys Ser Met Phe Val Lys Ile Lys Thr Leu Gly Ile 165 170
175 Gly Ala Phe Gly Glu Val Cys Leu Ala Cys Lys Val Asp Thr His Ala
180 185 190 Leu Tyr Ala Met Lys Thr Leu Arg Lys Lys Asp Val Leu Asn
Arg Asn 195 200 205 Gln Val Ala His Val Lys Ala Glu Arg Asp Ile Leu
Ala Glu Ala Asp 210 215 220 Asn Glu Trp Val Val Lys Leu Tyr Tyr Ser
Phe Gln Asp Lys Asp Ser 225 230 235 240 Leu Tyr Phe Val Met Asp Tyr
Ile Pro Gly Gly Asp Met Met Ser Leu 245 250 255 Leu Ile Arg Met Glu
Val Phe Pro Glu His Leu Ala Arg Phe Tyr Ile 260 265 270 Ala Glu Leu
Thr Leu Ala Ile Glu Ser Val His Lys Met Gly Phe Ile 275 280 285 His
Arg Asp Ile Lys Pro Asp Asn Ile Leu Ile Asp Leu Asp Gly His 290 295
300 Ile Lys Leu Thr Asp Phe Gly Leu Cys Thr Gly Phe Arg Trp Thr His
305 310 315 320 Asn Ser Lys Tyr Tyr Gln Lys Gly Ser His Val Arg Gln
Asp Ser Met 325 330 335 Glu Pro Ser Asp Leu Trp Asp Asp Val Ser Asn
Cys Arg Cys Gly Asp 340 345 350 Arg Leu Lys Thr Leu Glu Gln Arg Ala
Arg Lys Gln His Gln Arg Cys 355 360 365 Leu Ala His Ser Leu Val Gly
Thr Pro Asn Tyr Ile Ala Pro Glu Val 370 375 380 Leu Leu Arg Lys Gly
Tyr Thr Gln Leu Cys Asp Trp Trp Ser Val Gly 385 390 395 400 Val Ile
Leu Phe Glu Met Leu Val Gly Gln Pro Pro Phe Leu Ala Pro 405 410 415
Thr Pro Thr Glu Thr Gln Leu Lys Val Ile Asn Trp Glu Asn Thr Leu 420
425 430 His Ile Pro Ala Gln Val Lys Leu Ser Pro Glu Ala Arg Asp Leu
Ile 435 440 445 Thr Lys Leu Cys Cys Ser Ala Asp His Arg Leu Gly Arg
Asn Gly Ala 450 455 460 Asp Asp Leu Lys Ala His Pro Phe Phe Ser Ala
Ile Asp Phe Ser Ser 465 470 475 480 Asp Ile Arg Lys His Pro Ala Pro
Tyr Val Pro Thr Ile Ser His Pro 485 490 495 Met Asp Thr Ser Asn Phe
Asp Pro Val Asp Glu Glu Ser Pro Trp Asn 500 505 510 Asp Ala Ser Glu
Gly Ser Thr Lys Ala Trp Asp Thr Leu Thr Ser Pro 515 520 525 Asn Asn
Lys His Pro Glu His Ala Phe Tyr Glu Phe Thr Phe Arg Arg 530 535 540
Phe Phe Asp Asp Asn Gly Tyr Pro Phe Arg Cys Pro Lys Pro Ser Gly 545
550 555 560 Ala Glu Ala Ser Gln Ala Glu Ser Ser Asp Leu Glu Ser Ser
Asp Leu 565 570 575 Val Asp Gln Thr Glu Gly Cys Gln Pro Val Tyr Val
580 585
* * * * *
References