Note: Descriptions are shown in the official language in which they were submitted.
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V 197 DNA AND POLYPEPT1DES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Ser. No.
60/068,725,
filed December 24, 1997, which is hereby incorporated by reference.
FIELD OF THE INVENTION
The invention is directed to purified and isolated V I97 polypeptides, 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 as
molecular weight
markers, the use of such polypeptides and fragmented peptides as controls for
peptide
fragmentation, and kits comprising these reagents.
BACKGROUND OF THE INVENTION
The discovery and identification of proteins is 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.
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 ofMicroorganisms 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. Patent 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.
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,
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2
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 fi-agmentation 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-I 106, 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. Patent 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).
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)).
When a peptide fingerprint of an unlrnown 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-SO15, 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.embl-
heiedelberg.de...deSearch/FR PeptideSearchForm.html), and ProFound (Internet
site:
www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow
the user to
specify the cleavage agent and the molecular weights of the fragmented
peptides within a
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3
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.
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. Patent
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 o, f'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).
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
suitable for use in peptide
fragmentation studies and in molecular weight measurements.
SUMMARY OF THE INVENTION
The invention aids in fulfilling this need in the art. The invention
encompasses an
isolated nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1 and
an isolated
nucleic acid molecule encoding the amino acid sequence of SEQ ID N0:2. 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
and isolated nucleic acid molecules encoding the amino acid sequence of SEQ ID
N0:2. Both
single-stranded and double-stranded RNA and DNA V 197 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 of V 197 encompassed by the invention. A
double-
stranded DNA probe allows the detection of nucleic acid molecules equivalent
to either strand of
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4
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 or an isolated nucleic
acid
molecule encoding the amino acid sequence of SEQ ID N0:2 under conditions of
moderate
stringency in 50% formamide and 6XSSC, at 42°C with washing conditions
of 60°C, O.SXSSC,
0.1 % SDS are encompassed by the invention.
The invention further encompasses isolated nucleic acid molecules derived by
in vitro
mutagenesis from SEQ ID NO:1. 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 as a result of the genetic code,
isolated nucleic acid
molecules that are allelic variants of human V 197 DNA, or a species homolog
of V 197 DNA.
The invention also encompasses recombinant vectors that direct the expression
of these nucleic
acid molecules and host cells transformed or transfected with these vectors.
The invention also encompasses isolated polypeptides encoded by these nucleic
acid
1 S molecules, including isolated polypeptides having a molecular weight of
approximately 17 kD as
determined by SDS-PAGE and isolated polypeptides in non-glycosylated form.
Isolated
polyclonal or monoclonal antibodies that bind to these polypeptides are
encompassed by the
invention. The invention further encompasses methods for the production of V
197 polypeptides
including culturing a host cell under conditions promoting expression and
recovering the
polypeptide from the culture medium. Especially, the expression of V 197
polypeptides in
bacteria, yeast, plant, and animal cells is encompassed by the invention.
In addition, assays utilizing V 197 polypeptides to screen for potential
inhibitors of
activity associated with V 197 polypeptide counter-structure molecules, and
methods of using
V 197 polypeptides as therapeutic agents for the treatment of diseases
mediated by V 197
polypeptide counter-structure molecules are encompassed by the invention.
Further, methods of
using V 197 polypeptides in the design of inhibitors thereof are also an
aspect of the invention.
The invention further encompasses the fragmented peptides produced from V 197
polypeptides by chemical or enzymatic treatment. In addition, forms of V197
polypeptide
molecular weight markers 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.
The invention also encompasses a method for the visualization of V 197
polypeptide
molecular weight markers and fragmented peptides thereof using
electrophoresis. The invention
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- 5
further includes a method for using V 197 polypeptide molecular weight markers
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 further encompasses
methods for
using V 197 polypeptides 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 V 197 polypeptides and fragmented peptides thereof as
controls for
establishing the extent of fragmentation of a protein sample.
Further encompassed by this invention are kits to aid the determination of
molecular
weights of a sample protein utilizing V 197 polypeptide molecular weight
markers, fragmented
peptides thereof, and forms of V 197 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
A cDNA encoding human V 197 polypeptide has been isolated and is disclosed in
SEQ
ID NO:1.
The nucleotide sequence of V 197 DNA is:
ATGGACCGGCTTAAACAGAAGTACCAGAAGGAAGCAAGAAAGACAGGTTC
CTCCACTTTGGCGGTGGCCCTCTGCTCGACGGTGCCTTCGCTGGCCCTGA
CATCCCTGCTGTGCCTGGGCTTCGCCCTCTGTGCCTCAGTCCCCATCCTC
CCTCTCCAGTACCTCACCTTCATCCTGCAAGTGATCAGCCGCTCCTTCCT
CTATGGGAGCAACGCGGCCTTCCTCACCCTTGCTTTCCCTTCAGAGCACT
TTGGCAAGCTCTTTGGGCTGGTGATGGCCTTGTCGGCTGTGGTGTCTCTG
CTCCAGTTCCCCATCTTCACCCTCATCAAAGGCTCCCTTCAGAATGACCC
ATTTTACGTGAATGTGATGTTCATGCTTGCCATTCTTCTGACATTCTTCC
ACCCCTTTCTGGTATATCGGGAATGCCGTACTTGGAAAGAAAGTCCCTCT
GCAATTGCATAG (SEQ 1D NO:1 ).
The amino acid sequence of V 197 polypeptide is:
MDRLKQKYQKEARKTGSSTLAVALCSTVPSLALTSLLCLGFALCASVPIL
PLQYLTFILQVISRSFLYGSNAAFLTLAFPSEHFGKLFGLVMALSAWSL
LQFPIFTLIKGSLQNDPFYVNVMFMLAILLTFFHPFLVYRECRTWKESPS
AIA (SEQ ID N0:2) .
This discovery of the cDNA encoding human V 197 polypeptide (SEQ 1D N0:2)
enables
construction of expression vectors comprising nucleic acid sequences encoding
V 197
polypeptides; host cells transfected or transformed with the expression
vectors; biologically
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6
active human V 197 polypeptide and V 197 molecular weight markers as isolated
and purified
proteins; and antibodies immunoreactive with V 197 polypeptides.
Nucleotide sequence similar to V 197 DNA was originally seen in the TIGR-HGI
database. However, the nucleotide sequence of V 197 DNA was generated by
sequencing EST
clones, and is not identical to the TIGR-HGI nucleotide sequence. V 197 was
not found to be
highly related to any other sequences in the Genbank databases and, therefore,
represents unique
DNA and protein sequences.
V I97 polypeptide is a type I single transmembrane protein with a short
cytoplasmic tail,
and is probably a growth factor. V 197 polypeptide has a 44 amino acid leader
sequence (amino
acids 1-44 of SEQ ID N0:2), a 73 amino acid extracellular domain containing 1
potential N-
linked glycosylation site (amino acids 45-117 of SEQ ID N0:2), a 21 amino acid
transmembrane
domain (amino acids 118-138 of SEQ ID N0:2), and a 15 amino acid cytoplasmic
domain
(amino acids 139-153 of SEQ ID N0:2).
V 197 RNA is expressed as a mRNA of approximately 3.0 kb, that was detected by
northern blot in a wide variety of tissues. V 197 mRNA is expressed in heart,
very weakly in
brain, placenta, lung, strongly in liver, skeletal muscle, kidney, pancreas,
spleen, thymus,
prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes,
stomach, strongly in
thyroid, spinal cord, lymph node, trachea, bone marrow, and weakly in adrenal
gland.
In one embodiment of this invention, the expression of recombinant V 197
polypeptides
can be accomplished utilizing fusion of sequences encoding V 197 polypeptides
to sequences
encoding another polypeptide to aid in the purification of V 197 polypeptides.
An example of
such a fusion is a fusion of sequences encoding a V 197 polypeptide to
sequences encoding the
product of the malE gene of the pMAL-c2 vector of New England Bialabs, 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 V 197 polypeptide after
purification. It is
understood of course that many different vectors and techniques can be used
for the expression
and purification of V 197 polypeptides and that this embodiment in no way
limits the scope of the
invention.
The insertion of DNA encoding the V I97 polypeptide 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 V 197 polypeptide. In addition, the preferred construction of the
insertion results in the
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7
fusion of the amino terminus of the V 197 polypeptide 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 V 197 DNA 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 V 197 polypeptide from the fused maltose binding protein
utilizing a simple
protease treatment as per the manufacturer's instructions. In this manner,
purified V 197
polypeptide can be obtained. Furthermore, such a constructed vector can be
easily modified
using known molecular biology techniques to generate additional fusion
proteins.
Another preferred embodiment of the invention is the use of V 197 polypeptides
as
molecular weight markers to estimate the apparent molecular weight of a sample
protein by gel
electrophoresis. An isolated and purified V 197 polypeptide molecular weight
marker according
to the invention has a molecular weight of approximately 16,986 Daltons in the
absence of
glycosylation. The V 197 polypeptide, together with a sample protein, can be
resolved by
denaturing polyacrylamide gel electrophoresis by conventional means (LJ. 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 V 197 polypeptide molecular weight marker
can be used as
a molecular weight marker in the estimation of the apparent molecular weight
of the sample
protein. The unique amino acid sequence of V 197 (8EQ ID N0:2) specifies a
molecular weight
of approximately 16,986 Daltons. Therefore, the V 197 polypeptide molecular
weight marker
serves 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 16,986
Daltons. The use of this polypeptide molecular weight marker allows an
increased accuracy in
the determination of apparent molecular weight of proteins that have apparent
molecular weights
close to 16,986 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 V 197
polypeptides and
that this embodiment in no way limits the scope of the invention.
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8
Another preferred embodiment of the invention is the use of V 197 fragmented
peptide
molecular weight markers, generated by chemical fragmentation of V 197
polypeptide, as
molecular weight markers to estimate the apparent molecular weight of a sample
protein by gel
electrophoresis. Isolated and purified V 197 polypeptide can be treated with
cyanogen bromide
under conventional conditions that result in fragmentation of the V 197
polypeptide molecular
weight marker by specific hydrolysis on the carboxyl side of the methionine
residues within the
V 197 polypeptide (E. Gross, Methods in Enz. 11:238-255, 1967). Due to the
unique amino acid
sequence of the V 197 polypeptide, the fragmentation of V I97 polypeptide
molecular weight
markers with cyanogen bromide generates a unique set of V 197 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.
The unique set of V 197 fragmented peptide molecular weight markers generated
by
treatment of V 197 polypeptide with cyanogen bromide comprises 3 fragmented
peptides of at
least 10 amino acids in size. The peptide encoded by amino acids 2-92 of SEQ
1D N0:2 has a
molecular weight of approximately 9,881 Daltons. The peptide encoded by amino
acids 93-123
of SEQ m N0:2 has a molecular weight of approximately 3,423 Daltons. The
peptide encoded
by amino acids 126-I53 of SEQ ID N0:2 has a molecular weight of approximately
3,307
Daltons.
Therefore, cleavage of the V 197 polypeptide by chemical treatment with
cyanogen
bromide generates a unique set of V 197 fragmented peptide molecular weight
markers. The
unique and known amino acid sequence of these V 197 fragmented peptides allows
the
determination of the molecular weight of these fragmented peptide molecular
weight markers. In
this particular case, V197 fragmented peptide molecular weight markers have
molecular weights
of approximately 9,881; 3,423; and 3,307 Daltons.
The V 197 fragmented peptide molecular weight markers, 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 a.crylamide
between 10-20%. Proteins on the gel can be visualized using a conventional
staining procedure.
The V 197 fragmented peptide molecular weight markers 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 V197 specifies a molecular weight of approximately
9,881; 3,423; and
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3,307 Daltons for the V 197 fragmented peptide molecular weight markers.
Therefore, the V 197
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
apparent molecular weights close to 9,881; 3,423; or 3,307 Daltons.
Consequently, the use of
these fragmented peptide molecular weight markers allows an increased accuracy
in the
determination of apparent molecular weight of proteins that have apparent
molecular weights
close to 9,881; 3,423; or 3,307 Daltons.
In a further embodiment, the sample protein and the V 197 polypeptide can be
simultaneously, but separately, treated with cyanogen bromide under
conventional conditions
that result in fragmentation of the sample protein and the V 197 polypeptide
by specific
hydrolysis on the carboxyl side of the methionine residues within the sample
protein and the
V 197 polypeptide. As described above, the V 197 fragmented peptide molecular
weight markers
generated by cleavage of the V197 polypeptide with cyanogen bromide have
molecular weights
of approximately 9,881; 3,423; and 3,307 Daltons.
The fragmented peptides from both the V 197 polypeptide and the 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%. Fragmented peptides on the gel can be visualized using a
conventional
staining procedure. The V 197 fragmented peptide molecular weight markers can
be used as
molecular weight markers in the estimation of the apparent molecular weight of
the fragmented
proteins derived from the sample protein. As discussed above, the V 197
fragmented peptide
molecular weight markers serve particularly well as molecular weight markers
for the estimation
of the apparent molecular weight of fragmented peptides that have apparent
molecular weights
close to 9,881; 3,423; or 3,307 Daltons. Consequently, the use of these V197
fragmented peptide
molecular weight markers allows an increased accuracy in the determination of
apparent
molecular weight of fragmented peptides that have apparent molecular weights
close to 9,881;
3,423; or 3,307 Daltons. The extent of fragmentation of the V 197 polypeptide
is further used as
a control to determine the conditions expected for complete fragmentation of
the sample pmtein.
It is understood of course that many chemicals could be used to fragment V 197
polypeptides and
that this embodiment in no way limits the scope of the invention.
In another embodiment, unique sets of V 197 fragmented peptide molecular
weight
markers can be generated from V 197 polypeptide using enzymes that cleave the
polypeptide at
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specific amino acid residues. Due to the unique nature of the amino acid
sequence of the V 197
polypeptide, cleavage at different amino acid residues will result in the
generation of different
sets of fragmented peptide molecular weight markers.
An isolated and purified V 197 polypeptide can be treated with Achromobacter
protease I
5 under conventional conditions that result in fragmentation of the V 197
polypeptide by specific
hydrolysis on the carboxyl side of the lysine residues within the V 197
polypeptide (T. Masaki et
al., Biochim. Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim.
Biophys. Acta 660:51-55,
1981 ). Due to the unique amino acid sequence of the V 197 polypeptide, the
fragmentation of
V 197 polypeptide molecular weight markers with Achromobacter protease I
generates a unique
10 set of V 197 fragmented peptide molecular weight markers. The distribution
of lysine residues
determines the number of amino acids in each peptide and the unique amino acid
composition of
each peptide determines its molecular weight.
The unique set of V 197 fragmented peptide molecular weight markers generated
by
treatment of V 197 polypeptide with Achromobacter protease I comprises 3
fragmented peptides
of at least 10 amino acids in size. Although 3 fragmented peptides are
generated with this
enzyme treatment of the V 197 polypeptide, similar to the generation of 3
fragmented peptides
with cyanogen bromide treatment of the V 197 polypeptide, the sizes of the
fragmented peptides
clearly illustrate that the sizes of the fragmented peptide molecular weight
markers will vary
depending upon the fragmentation treatment utilized to fragment the V 197
polypeptide. Both the
size and number of these fragments are dictated by the amino acid sequence of
the V 197
polypeptide. Consequently, the number of fragmented peptides will also vary
depending upon
the fragmentation treatment utilized to fragment the V 197 polypeptide.
The peptide encoded by amino acids 15-86 of SEQ >D N0:2 has a molecular weight
of
approximately 7,549 Daltons. The peptide encoded by amino acids 87-110 of SEQ
11'? N0:2 has
a molecular weight of approximately 2,619 Daltons. The peptide encoded by
amino acids 111-
146 of SEQ ID N0:2 has a molecular weight of approximately 4,395 Daltons.
Therefore, cleavage of the V 197 polypeptide by enzymatic treatment with
Achromobacter
protease I generates a unique set of V 197 fragmented peptide molecular weight
markers. The
unique and known amino acid sequence of these fragmented peptides allows the
determination of
the molecular weight of these V I97 fragmented peptide molecular weight
markers. In this
particular case, these V 197 fragmented peptide molecular weight markers have
molecular
weights of approximately 7,549; 2,619; and 4,395 Daltons.
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Once again, the V 197 fragmented peptide molecular weight markers, 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 V 197 fragmented peptide molecular weight
markers can
be used as molecular weight markers in the estimation of the apparent
molecular weight of the
sample protein. The V 197 fragmented peptide molecular weight markers serve
particularly well
as molecular weight markers for the estimation of the apparent molecular
weight of proteins that
have apparent molecular weights close to 7,549; 2,619; or 4,395 Daltons. The
use of these
fragmented peptide molecular weight markers allows an increased accuracy in
the determination
of apparent molecular weight of proteins that have apparent molecular weights
close to 7,549;
2,619; or 4,395 Daltons.
In another embodiment, the sample protein and the V 197 polypeptide can be
simultaneously, but separately, treated with Achromobacter protease I under
conventional
conditions that result in fragmentation of the sample protein and the V 197
polypeptide by
specific hydrolysis on the carboxyl side of the lysine residues within the
sample protein and the
V I 97 polypeptide. The V 197 fragmented peptide molecular weight markers and
the fragmented
peptides derived from the sample protein are 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 I O-20%. Fragmented peptides
on the gel can
be visualized using a conventional staining procedure. The V 197 fragmented
peptide molecular
weight markers can be used as molecular weight markers in the estimation of
the apparent
molecular weight of the sample protein. The V 197 fragmented peptide molecular
weight
markers serve particularly well as molecular weight markers for the estimation
of the apparent
molecular weight of fragmented peptides that have apparent molecular weights
close to 7,549;
2,619; or 4,395 Daltons. The use of these V 197 fragmented peptide molecular
weight markers
allows an increased accuracy in the determination of apparent molecular weight
of fragmented
peptides that have apparent molecular weights close to 7,549; 2,619; or 4,395
Daltons. The
extent of fragmentation of the V197 polypeptide is further used as a control
to determine the
conditions expected for complete fragmentation of the sample protein. It is
understood of course
that many enzymes could be used to fragment V 197 polypeptides and that this
embodiment in no
way limits the scope of the invention.
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12
In another embodiment, monoclonal and polyclonal antibodies against V 197
polypeptides
can be generated. Balb/c mice can be injected intraperitoneally on two
occasions at 3 week
intervals with 10 ~,g of isolated and purified V197 polypeptide or peptides
based on the amino
acid sequence of V 197 polypeptides in the presence of RIBI adjuvant (RIBI
Corp., Hamilton,
Montana). Mouse sera are then assayed by conventional dot blot technique or
antibody capture
(ABC) to determine which animal is best to fuse. Three weeks later, mice are
given an
intravenous boost of 3 pg of the V 197 polypeptide or peptides, suspended in
sterile PBS. Three
days later, mice are sacrificed and spleen cells fused with Ag8.653 myeloma
cells (ATCC)
following established protocols. Briefly, Ag8.653 cells are washed several
times in serum-free
media and fused to mouse spleen cells at a ratio of three spleen cells to one
myeloma cell. The
fusing agent is 50% PEG: 10% DMSO (Sigma). Fusion is plated out into twenty 96-
well flat
bottom plates (Coming) containing HAT supplemented DMEM media and allowed to
grow for
eight days. Supernatants from resultant hybridomas are collected and added to
a 96-well plate
for 60 minutes that is first coated with goat anti-mouse Ig. Following washes,
'ZSI-V 197
polypeptide or peptides are added to each well, incubated for 60 minutes at
room temperature,
and washed four times. Positive wells can be subsequently detected by
autoradiography at -70°C
using Kodak X-Omat S film. Positive clones can be grown in bulk culture and
supernatants are
subsequently purified over a Protein A column (Pharmacia). It is understood of
course that many
techniques could be used to generate antibodies against V 197 polypeptides and
fragmented
peptides thereof and that this embodiment in no way limits the scope of the
invention.
In another embodiment, antibodies generated against V 197 and fragmented
peptides
thereof can be used in combination with V 197 polypeptide or fragmented
peptide molecular
weight markers 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. V 197
polypeptide or fi~agmented peptide molecular weight markers 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.
Polypeptides on the membrane can be visualized using two different methods
that allow a
discrimination between the sample protein and the molecular weight markers. V
197 polypeptide
or fragmented peptide molecular weight markers can be visualized using
antibodies generated
against these markers and conventional immunoblotting techniques. This
detection is performed
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13
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 V
197 polypeptide
fragments, 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
V 197 polypeptides and fragments can be readily determined using conventional
techniques.
The sample protein is visualized using a conventional staining procedure. The
molar
excess of sample protein to V 197 polypeptide or fragmented peptide molecular
weight markers is
such that the conventional staining procedure predominantly detects the sample
protein. The
level of V 197 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 V 197 polypeptide molecular weight markers is
between 2 and
100,000 fold. More preferably, the preferred molar excess of sample protein to
V 197
polypeptide molecular weight markers is between 10 and 10,000 fold and
especially between 100
and 1;000 fold.
The V197 polypeptide or fragmented peptide molecular weight markers 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. The V 197 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 V 197
polypeptide or fragmented peptide molecular weight markers. The ability to
simultaneously
resolve the V 197 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.
In another embodiment, V 197 polypeptide or fragmented peptide molecular
weight
markers 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
CA 02316016 2000-06-21
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14
fragmented peptides thereof using V 197 polypeptide molecular weight markers
and peptide
fragments thereof and that this embodiment in no way limits the scope of the
invention.
V 197 polypeptide molecular weight markers encompassed by invention can have
variable
molecular weights, depending upon the host cell in which they are expressed.
Glycosylation of
V 197 polypeptide molecular weight markers and peptide fragments thereof in
various cell types
can result in variations of the molecular weight of these markers, depending
upon the extent of
modification. The size of V 197 polypeptide molecular weight markers can be
most
heterogeneous with fragments of V I 97 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.
The interaction between V 197 and its counter-structure enables screening for
small
molecules that interfere with the V 197/V 197 counter-structure association
and inhibit activity of
V I 97 or its counter-structure. For example, the yeast two-hybrid system
developed at SUNY
(described in U.S. Patent No. 5,283,173 to Fields et al.) can be used to
screen for inhibitors of
V 197 as follows. V 197 and its counter-structure, or portions thereof
responsible for their
interaction, can be fused to the Gal4 DNA binding domain and Gal 4
transcripdonal activation
domain, respectively, and introduced into a strain that depends on Gal4
activity for growth on
plates lacking histidine. Compounds that prevent growth can be screened in
order to identify IL-
1 inhibitors. Alternatively, the screen can be modified so that V 197/V 197
counter-structure
interaction inhibits growth, so that inhibition of the interaction allows
growth to occur. Another,
in vitro, approach to screening for V 197 inhibition would be to immobilize
one of the
components (either V 197 or its counter-structure) in wells of a microtiter
plate, and to couple an
easily detected indicator to the other component. An inhibitor of the
interaction is identified by
the absence of the detectable indicator from the well.
In addition, V 197 polypeptides according to the invention are useful for the
structure-
based design of V I97 inhibitor. Such a design would comprise the steps of
determining the
three-dimensional structure of such the V 197 polypeptide, analyzing the three-
dimensional
structure for the likely binding sites of substrates, synthesizing a molecule
that incorporates a
predictive reactive site, and determining the inhibiting activity of the
molecule.
Antibodies immunoreactive with V 197 polypeptides, and in particular,
monoclonal
antibodies against V 197 polypeptides, are now made available through the
invention. Such
CA 02316016 2000-06-21
WO 99133984 PCT/US98IZ7627
antibodies can be useful for inhibiting V 197 polypeptide activity in vivo and
for detecting the
presence of V 197 polypeptides in a sample.
As used herein, the term "V 197 polypeptides" refers to a genus of
polypeptides that
further encompasses proteins having the amino acid sequence 1-153 of SEQ ID
N0:2, as well as
S those proteins having a high degree of similarity (at least 90% identity)
with such amino acid
sequences and which proteins are biologically active. In addition, V I97
polypeptides refers to
the gene products of the nucleotides 1-462 of SEQ ID NO:1.
The isolated and purified V 197 polypeptide according to the invention has a
molecular
weight of approximately 16,986 Daltons in the absence of glycosylation. It is
understood that the
10 molecular weight of V 197 polypeptides can be varied by fusing additional
peptide sequences to
both the amino and carboxyl terminal ends of V I97 polypeptides. Fusions of
additional peptide
sequences at the amino and carboxyl terminal ends of V 197 polypeptides can be
used to enhance
expression of V 197 polypeptides or aid in the purification of the protein.
It is understood that fusions of additional peptide sequences at the amino and
carboxyl
15 terminal ends of V 197 polypeptides will alter some, but usually not all,
of the fragmented
peptides of V 197 polypeptides generated by enzymatic or chemical treatment.
It is understood that mutations can be introduced into V 197 polypeptides
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 V 197
polypeptides upon fragmentation
with the specific enzyme or chemical procedure.
The term "isolated and purified" as used herein, means that the V 197
polypeptide
molecular weight markers or fragments thereof 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 V 197 polypeptide molecular
weight markers or
fragments thereof 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 V197 polypeptides or fragments thereof can be used as
molecular weight
markers. The term "purified" refers to either the "isolated and purified" form
of V 197
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16
polypeptides or the "substantially purified" form of V 197 polypeptides, as
both are described
herein.
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, NY (1989)). Such sequences are preferably
provided 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.
A V 197 polypeptide "variant" as referred to herein means a polypeptide
substantially
homologous to native V 197 polypeptides, but which has an amino acid sequence
different from
that of native V 197 polypeptides (human, marine or other mammalian species)
because of one or
more deletions, insertions or substitutions. The variant amino acid sequence
preferably is at least
80% identical to a native V 197 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 (LJWGCG). 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.
Variants can comprise conservatively substituted sequences, meaning that a
given amino
acid residue is replaced by a residue having similar physiochemical
characteristics. Examples of
conservative substitutions include substitution of one aliphatic residue for
another, such as Ile,
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17
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 GIn and Asn. Other such conservative
substitutions, for
example, substitutions of entire regions having similar hydrophobicity
characteristics, are well
known. Naturally occurring V 197 variants are also encompassed by the
invention. Examples of
such variants are proteins that result from alternate mRNA splicing events or
from proteolytic
cleavage of the V 197 polypeptides. 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 V 197
polypeptides (generally
from 1-5 terminal amino acids).
As stated above, the invention provides isolated and purified, or homogeneous,
V 197
polypeptides, both recombinant and non-recombinant. Variants and derivatives
of native V 197
polypeptides that can be used as molecular weight markers can be obtained by
mutations of
nucleotide sequences coding for native V 197 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 Iigation to fragments of the native
sequence. Following
ligation, the resulting reconstructed sequence encodes an analog having the
desired amino acid
insertion, substitution, or deletion.
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 Walden 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. Patent Nos. 4,518,584 and
4,737,462, all of
which are incorporated by reference.
V 197 polypeptides can be modified to create V 197 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 V 197 polypeptides can be prepared by linking the chemical
moieties to functional
groups on V 197 polypeptide amino acid side chains or at the N-terminus or C-
terminus of a
V 197 polypeptide or the extracellular domain thereof. Other derivatives of V
197 polypeptides
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18
within the scope of this invention include covalent or aggregative conjugates
of V 197
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 a-factor leader of
Saccharomyces) at
S the N-terminus of a V 197 polypeptide. 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.
V I 97 polypeptide conjugates can comprise peptides added to facilitate
purification and
identification of V 197 polypeptides. Such peptides include, for example, poly-
His or the
I O antigenic identification peptides described in U.S. Patent No. S,OI 1,912
and in Hopp et al.,
BiolTechnology 6:1204, 1988.
The invention further includes V 197 polypeptides with or without associated
native-
pattem glycosylation. V 197 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 V197
15 polypeptide in molecular weight and glycosylation pattern, depending upon
the choice of
expression system. Expression of V 197 polypeptides in bacterial expression
systems, such as E.
coli, provides non-glycosylated molecules. GIycosyl groups can be removed
through
conventional methods, in particular those utilizing glycopeptidase. In
general, glycosylated
V 197 polypeptides can be incubated with a molar excess of glycopeptidase
(Boehringer
20 Mannheim).
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 V 197 polypeptide
extracellular
domain can be modified to preclude glycosylation, allowing expression of a
reduced
25 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
30 is replaced by a different amino acid, for example, is Buff cient to
inactivate an N-glycosylation
site. Known procedures for inactivating N-glycosylation sites in proteins
include those described
in U.S. Patent 5,071,972 and EP 276,846, hereby incorporated by reference.
CA 02316016 2000-06-21
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19
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.
ICEX2 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.
The invention fiu~ther encompasses isolated fragments and oligonucleotides
derived from
the nucleotide sequence of SEQ ID NO:1. The invention also encompasses
polypeptides
encoded by these fragments and oligonucleotides.
Nucleic acid sequences within the scope of the invention include isolated DNA
and RNA
sequences that hybridize to the native V 197 nucleotide sequences disclosed
herein under
conditions of moderate or severe stringency, and which encode V 197
polypeptides. 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 SX SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization
conditions of
50% formamide, 6X SSC at 42°C (or other similar hybridization solution,
such as Stark's
solution, in 50% formamide at 42°C), and washing conditions of about
60°C, O.SX SSC, 0.1%
SDS. Conditions of high stringency are defined as hybridization conditions as
above, and with
washing at 68°C, 0.2X 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.
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 1D
NO:1 and
still encode a V 197 polypeptide having the amino acid sequence of SEQ ID
N0:2. 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.
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- 20
The invention thus provides equivalent isolated DNA sequences encoding V 197
polypeptides, selected from: (a) DNA derived from the coding region of a
native mammalian
V197 gene; (b) cDNA comprising the nucleotide sequence 1-4.62 of SEQ ID NO:1;
(c) DNA
capable of hybridization to a DNA of (a) under conditions of moderate
stringency and which
encodes V 197 polypeptides; and (d) DNA which is degenerate as a result of the
genetic code to a
DNA defined in (a), (b) or (c) and which encodes V 197 polypeptides. V 197
polypeptides
encoded by such DNA equivalent sequences are encompassed by the invention.
DNA that is equivalent to the DNA sequence of SEQ ID NO:1 will hybridize under
moderately stringent conditions to the double-stranded native DNA sequence
that encode
polypeptides comprising amino acid sequences of 1-153 of SEQ ID N0:2. Examples
of V197
polypeptides encoded by such DNA, include, but are not limited to, V 197
polypeptide fragments
and V 197 polypeptides comprising inactivated N-glycosylation site(s),
inactivated protease
processing site(s), or conservative amino acid substitution(s), as described
above. V 197
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 are also encompassed.
V 197 polypeptide-binding proteins, such as the anti-V 197 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
V 197 polypeptides on
their surface. Adherence of V 197 polypeptide-binding proteins to a solid
phase contacting
surface can be accomplished by any means, for example, magnetic microspheres
can be coated
with V197 polypepdde-binding proteins and held in the incubation vessel
through a magnetic
field. Suspensions of cell mixtures are contacted with the solid phase that
has V 197 polypeptide-
binding proteins thereon. Cells having V 197 polypeptides on their surface
bind to the fixed
V 197 polypeptide-binding protein and unbound cells then are washed away. This
affinity-
binding method is useful for purifying, screening or separating such V 197
polypeptide-
expressing cells from solution. Methods of releasing positively selected cells
from the solid
phase are lrnown 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.
Alternatively, mixtures of cells suspected of containing V 197 polypeptide-
expressing
cells first can be incubated with a biotinylated V 197 polypeptide-binding
protein. Incubation
periods are typically at least one hour in duration to ensure sufficient
binding to V 197
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- 21
polypeptides. 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 V 197
polypeptide-binding cells to the beads. Use of avidin-coated beads is known in
the art. See
Berenson, et al. J. Cell. Biochem., lOD:239 (1986). Wash of unbound material
and the release of
the bound cells is performed using conventional methods.
In the methods described above, suitable V 197 polypeptide-binding proteins
are anti-
V 197 polypeptide antibodies, and other proteins that are capable of high-
amity binding of V 197
polypeptides. A preferred V 197 polypeptide-binding protein is an anti-V 197
polypeptide
monoclonal antibody.
V 197 polypeptides can 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 V 197 polypeptides. In one embodiment of the invention,
a V 197
polypeptide dimer is created by fusing V 197 polypeptides to the Fc region of
an antibody (e.g.,
IgGl) in a manner that does not interfere with biological activity of V197
polypeptides. The Fc
polypeptide preferably is fused to the C-terminus of a soluble V 197
polypeptide (comprising
only the extracellular domain). General preparation of fusion proteins
comprising heterologous
poiypeptides 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 V 197
polypeptide:Fc fusion protein is inserted into an appropriate expression
vector. V 197
polypeptide:Fc fusion proteins are allowed to assemble much like antibody
molecules,
whereupon interchain disulfide bonds form between Fc polypeptides, yielding
divalent V 197
polypeptides. If fusion proteins are made with both heavy and light chains of
an antibody, it is
possible to form a V197 polypeptide oligomer with as many as four V197
polypeptides
extracellular regions. Alternatively, one can link two soluble V 197
polypeptide domains with a
peptide linker.
Recombinant expression vectors containing a nucleic acid sequence encoding V I
97
polypeptides can be prepared using well known methods. The expression vectors
include a V 197
DNA sequence 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
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22
initiation and termination. Nucleotide sequences are "operably linked" when
the regulatory
sequence functionally relates to the V 197 DNA sequence. Thus, a promoter
nucleotide sequence
is operably linked to a V 197 DNA sequence if the promoter nucleotide sequence
controls the
transcription of the V 197 DNA sequence. 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.
In addition, sequences encoding appropriate signal peptides that are not
naturally
associated with V 197 polypeptides can be incorporated into expression
vectors. For example, a
DNA sequence for a signal peptide (secretory leader) can be fused in-frame to
the V 197
nucleotide sequence so that the V 197 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 V 197 polypeptide. The signal peptide
can be cleaved
from the V 197 polypeptide upon secretion of V 197 polypeptide from the cell.
Suitable host cells for expression of V 197 polypeptides 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 V 197 polypeptides using RNAs derived from DNA
constructs
disclosed herein.
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 V 197
polypeptide 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 V I97 polypeptide.
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 piasmids such as the cloning vector pBR322
(ATCC
37017). pBR322 contains genes for ampicillin and tetracycline resistance and
thus provides
CA 02316016 2000-06-21
WO 99/33984 PCT/US98IZ7627
23
simple means for identifying transformed cells. To construct en expression
vector using
pBR322, an appropriate promoter and a V 197 DNA sequence are inserted into the
pBR322
vector. Other commercially available vectors include, for example, pKK223-3
(Pharmacia Fine
Chemicals, Uppsaia, Sweden) and pGEMl (Promega Biotec, Madison, WI, 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, MA,
USA).
Promoter sequences commonly used for recombinant prokaryotic host cell
expression
vectors include (3-lactamase (penicillinase), lactose promoter system (Chang
et al., Nature
27,f: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 ~
PL promoter and a
cI857ts thermolabile repressor sequence. Plasmid vectors available from the
American Type
IS Culture Collection, which incorporate derivatives of the ~. PL promoter,
include plasmid pHUB2
(resident in E. toll strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. toll
RRl (ATCC
53082)).
V 197 DNA may be cloned in-frame into the multiple cloning site of an ordinary
bacterial
expression vector. Ideally the vector would contain 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 may be
boosted by incorporation of colons encoding a fusion partner (such as
hexahistidine) between
the promoter and the gene of interest. The resulting "expression plasnud" may
be propagated in
a variety of strains of E. toll.
For expression of the recombinant pmtein, 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 x G for
20 minutes at 4°C.
For recovery of the expressed protein, the peileted cells may be resuspended
in ten
volumes of 50 mM Tris-HCl (pH 8)/1 M NaCI and then passed two or three times
through a
CA 02316016 2000-06-21
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24
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 x G for 20 minutes, 4°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-HCI (pH 8)/8
M urea/ 0.1
M DTT. Any material that cannot be dissolved is removed by centrifugation
(10,000 x G for 20
minutes, 20°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 SO mM Tris-HCl (pH 8)/5 mM CaCI~/5 mM Zn(OAc)_/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.
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.
V 197 polypeptides 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 lactic, or
Kluyveromyces, can also be employed. Yeast vectors will often contain an
origin of replication
sequence from a 2p 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., BiolTechnology, 8:135-139 (1990). Another alternative is
the glucose-
repressible ADH2 promoter described by Russell et al. (J. Biol. Chem.
258:2674, 1982) and
CA 02316016 2000-06-21
WO 99133984 PCT/US98/27627
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
(Amp gene and origin of replication) into the above-described yeast vectors.
The yeast a-factor leader sequence can be employed to direct secretion of a V
197
5 polypeptide. The a-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. Patent 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
10 restriction sites. This will facilitate fusion of the leader sequence to
the structural gene.
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+ transformants in a selective medium, wherein the
selective medium
consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, IO
~cg/ml adenine, and
I S 20,ug/ml uracil.
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 ~ug/ml
adenine and 80 ,ug/ml uracil. Derepression of the ADH2 promoter occurs when
glucose is
20 exhausted from the medium.
Mammalian or insect host cell culture systems could also be employed to
express
recombinant V 197 polypeptides. Baculovirus systems for production of
hetemlogous proteins in
insect cells are reviewed by Luckow and Summers, BiolTechnology 6:47 (1988).
Established
cell lines of mammalian origin also can be employed. Examples of suitable
mammalian host cell
25 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-IBBNA-I 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).
Established methods for introducing DNA into mammalian cells have been
described
(Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69).
Additional protocols
using commercially available reagents, such as Lipofectamine (GibcoBRL) or
Lipofectamine-
CA 02316016 2000-06-21
WO 99133984 PCTIUS98/27627
26
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 resistance to cytotoxic drugs as a selection method. Kaufinan
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 (LJrlaub 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
I0 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 G4I8 and hygromycin B. Cells harboring the
vector can be
selected on the basis of resistance to these compounds.
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 (Hers et al.,
Nature 273:113, 1978; Kaufinan, Meth. in Enzymology, 1990). Smaller or larger
SV40
fragments can also be used, provided the approximately 250 by sequence
extending from the
Hind III site toward the Bgl I site located in the SV40 viral origin of
replication site is included.
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 dicistmnic 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
CA 02316016 2000-06-21
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27
gene for a selectable marker (eg. DHFR) has been Shawn to improve
transfectability of the host
and expression of the heterologous cDNA (Kaufinan, Meth. in Enrymology, 1990).
Exemplary
expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by
Mosser et al.,
Biotechniques 22:150-161, 1997, and p2ASI described by Morris et al., Animal
Cell Technology,
1997, pp. 529-534.
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 C127 marine 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 Serial No. 071701,415, 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 United States Patent 4,965,195; the signal sequence for
IL-2 receptor
described in Cosirian 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. Patent
4,968,607; and the type
H IL-1 receptor signal peptide described in EP 460,846.
An isolated and purified V 197 polypeptide molecular weight marker according
to the
invention can be produced by recombinant expression systems as described above
or purified
from naturally occurring cells. V 197 polypeptides can be substantially
purified, as indicated by a
single protein band upon analysis by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
One process for producing V 197 polypeptides comprises culturing a host cell
transformed
with an expression vector comprising a DNA sequence that encodes a V 197
polypeptide under
conditions sufficient to promote expression of the V 197 polypeptide. V 197
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
CA 02316016 2000-06-21
WO 99133984 PCT1US98/27627
28
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
carboxymethyi 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
fiuther purify V 197 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.
It is possible to utilize an affinity column comprising a V 197 polypeptide-
binding
protein, such as a monoclonal antibody generated against V 197 polypeptides,
to affinity-purify
expressed V 197 polypeptides. V 197 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.
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.
Transformed yeast host cells are preferably employed to express V 197
polypeptides 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.
V 197 polypeptide molecular weight markers can be analyzed by methods
including
sedimentation, gel electrophoresis, chromatography, and mass spectrometry. V
197 polypeptides
can serve as molecular weight markers using such analysis techniques to assist
in the
CA 02316016 2000-06-21
WO 99133984 PCT/US98/Z7627
- 29
determination of the molecular weight of a sample protein. A molecular weight
determination of
the sample protein assists in the identification of the sample protein.
V 197 polypeptides 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 fiuther include further
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,
Arginylendopeptidase, Achrombobacter 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. Asparaginylendopeptidase can cleave
specifically on
the carboxyl side of the asparagine residues present within V 197
polypeptides.
Arginylendopeptidase can cleave specifically on the carboxyl side of the
arginine residues
present within V 197 polypeptides. Achrombobacter protease I can cleave
specifically on the
carboxyl side of the lysine residues present within V197 polypeptides
(Sakiyama and Nakat, U.S.
Patent 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 V197 polypeptides.
Staphlococcus aureus V8
protease can cleave specifically on the carboxyl side of the aspartic and
glutamic acid residues
present within V197 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 V 197 polypeptides. Endoproteinase Lys-C can cleave
specifically on the carboxyl
side of the lysine residues present within V 197 polypeptides. Other enzymatic
and chemical
treatments can likewise be used to specifically fragment V I97 polypeptides
into a unique set of
specific peptide molecular weight markers.
The resultant fragmented peptides can be analyzed by methods including
sedimentation,
electrophoresis, chromatograpy, and mass spectrometry. The fragmented peptides
derived from
V 197 polypeptides 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. V 197
fragmented peptide
molecular weight markers are preferably between 10. and 152 amino acids in
size. More
preferably, V 197 fragmented peptide molecular weight markers are between 10
and 100 amino
CA 02316016 2000-06-21
WO 99/33984 PCT/US98/27627
acids in size. Even more preferable are V 197 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 V 197 fragmented peptide molecular weight markers between
10 and 20
amino acids in size.
S Furthermore, analysis of the progressive fragmentation of V 197 polypeptides
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 V 197
polypeptide and sample protein under identical conditions can allow for a
direct comparison of
10 the extent of fragmentation. Conditions that result in the complete
fragmentation of V 197
polypeptide can also result in complete fragmentation of the sample protein.
In addition, V 197 polypeptides and fragmented peptides thereof 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
15 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 )). V 197
polypeptides and fragmented peptides thereof can be used in such analyses as
markers to assist in
20 the determination of both the isoelectric point and molecular weight of a
sample protein or
fragmented peptide.
Kits to aid in the determination of apparent molecular weight and isoelectric
point of a
sample protein can be assembled from V 197 polypeptides and peptide fragments
thereof. Kits
also serve to assess the degree of fragmentation of a sample protein. The
constituents of such
25 kits can be varied, but typically contain V 197 polypeptide and fragmented
peptide molecular
weight markers. Also, such kits can contain V 197 polypeptides wherein a site
necessary for
fragmentation has been removed. Furthermore, the kits can contain reagents for
the specific
cleavage of V 197 and the sample protein by chemical or enzymatic cleavage.
Kits can further
contain antibodies directed against V 197 polypeptides or fragments thereof.
30 Antisense or sense oligonucleotides comprising a single-stranded nucleic
acid sequence
(either RNA or DNA) capable of binding to a target V 197 mRNA sequence
(forming a duplex)
or to the V 197 sequence in the double-stranded DNA helix {forming a triple
helix) can be made
CA 02316016 2000-06-21
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- 31
according to the invention. Antisense or sense oligonucleotides, according to
the present
invention, comprise a fragment of the coding region of V 197 cDNA {SEQ ID NO:1
). 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.
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 V 197 polypeptides. Antisense or sense oligonucleotides
further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or other
sugar linkages, such
as those described in W091/06629) 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.
Antisense or sense oligonucleotides can be introduced into a cell containing
the target
nucleic acid sequence by any gene transfer method, including, for example,
CaP04-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
marine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double
copy
vectors designated DCTSA, DCTSB and DCTSC (see PCT Application US 90/02656).
CA 02316016 2000-06-21
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32
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 Iigand 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.
Alternatively, 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.
Isolated and purified V 197 polypeptides or a fragment thereof can also be
useful itself as
a therapeutic agent in inhibiting IL-1 and TNF signaling. V197 polypeptides
are introduced into
the intracellular environment by well-known means, such as by encasing the
pmtein in liposomes
or coupling it to a monoclonal antibody targeted to a specific cell type.
V 197 DNA, V 197 polypeptides, and antibodies against V 197 polypeptides 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 Hatbor
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 constituitive and transient expression of V 197 RNA or
polypeptides. V 197 DNA can
be used to determine the chromosomal location of V 197 DNA and to map genes in
relation to
this chromosomal location. V 197 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. V 197 DNA can be further used to identify
additional genes
related to V 197 DNA and to establish evolutionary trees based on the
comparison of sequences.
V 197 DNA and polypeptides can be used to select for those genes or proteins
that are
homologous to V 197 DNA or polypeptides, through positive screening procedures
such as
Southern blotting and immunoblotting and through negative screening procedures
such as
subtraction.
V 197 polypeptides can also be used as a reagent to identify {a) any protein
that V 197
polypeptide regulates, and (b) other proteins with which it might interact. V
197 polypeptides
CA 02316016 2000-06-21
WO 99133984 PCTIUS98/Z7627
- 33
could be used by coupling recombinant protein to an affinity matrix, or by
using them as a bait in
the 2-hybrid system. V 197 polypeptides and fragments thereof can be used as
reagents in the
study of the IL-1 signaling pathway as a reagent to block IL-1 signaling.
When used as a therapeutic agent, V 197 polypeptides can be formulated into
pharmaceutical compositions according to known methods. V 197 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-HCI, 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
V I97
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,
I 5 rate of in vivo release, and rate of in vivo clearance of V 197
polypeptides.
Within an aspect of the invention, V 197 polypeptides, and peptides based on
the amino
acid sequence of V 197, can be utilized to prepare antibodies that
specifically bind to V I97
polypeptides. The term "antibodies" is meant to include polyclonai 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 V 197
polypeptides with a ICe of greater than or equal to about 10' M''. 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).
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 V 197 polypeptides, or a peptide based
on the amino acid
sequence of V 197 polypeptides that is appropriately conjugated, is
administered to the host
animal typically through parenteral injection. The immunogenicity of V 197
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 V 197 polypeptides. Examples of various assays useful for such
determination include those
described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor
CA 02316016 2000-06-21
WO 99/33984 PCTNS98/27627
34
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. Patent Nos. 4,376,110
and 4,486,530.
Monoclonal antibodies can be readily prepared using well-known procedures, see
for
example, the procedures described in U.S. Patent 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, McKearn, and Bechtol (eds.), 1980. Briefly, the host
animals, such as
mice are injected intraperitoneally at least once, and preferably at least
twice at about 3 week
intervals with isolated and purified V 197 polypeptides or conjugated V 197
polypeptides,
optionally in the presence of adjuvant. Mouse sera are then assayed by
conventional dot blot
technique or antibody capture (ABC) to determine which animal is best to fuse.
Approximately two to three weeks later, the mice are given an intravenous
boost of V 197
polypeptides or conjugated V 197 polypeptides. 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).
Fusion is plated out
into plates containing media that allows for the selective growth of the fused
cells. The fused
cells can then be allowed to grow for approximately eight days. Supernatants
from resultant
hybridomas are collected and added to a plate that is first coated with goat
anti-mouse Ig.
Following washes, a label, such as, 'ZSI-V 197 polypeptides is added to each
well followed by
incubation. Positive wells can be subsequently detected by autoradiography.
Positive clones can
be grown in bulk culture and supernatants are subsequently purified over a
Protein A column
(Pharmacia).
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).
CA 02316016 2000-06-21
WO 99/33984 PCTNS98/Z7627
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
V 197 polypeptides are also encompassed by the invention.
5 Once isolated and purified, the antibodies against V 197 polypeptides can be
used to
detect the presence of V 197 polypeptides in a sample using established assay
protocols. Further,
the antibodies of the invention can be used therapeutically to bind to V 197
polypeptides and
inhibit its activity in vivo.
The purified V 197 polypeptides according to the invention will facilitate the
discovery of
10 inhibitors of V I97 polypeptides. The use of a purified V I 97 polypeptide
in the screening of
potential inhibitors thereof is important and can eliminate or reduce the
possibility of interfering
reactions with contaminants.
In addition, V 197 polypeptides can be used for structure-based design of V
197
polypeptide-inhibitors. Such structure-based design is also known as "rational
drug design."
15 The V197 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 V 197 polypeptide structural information in molecular
modeling software
systems to assist in inhibitor design and inhibitor-V 197 polypeptide
interaction is also
encompassed by the invention. Such computer-assisted modeling and drug design
can utilize
20 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
25 particular method of the invention comprises analyzing the three
dimensional structure of V 197
polypeptides for likely binding sites of substrates, synthesizing a new
molecule that incorporates
a predictive reactive site, and assaying the new molecule as described above.
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
30 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.
CA 02316016 2000-06-21
WO 99/33984 PCT/US98I27627
1
SEQUENCE LISTING
<110> Lyman, Stewart
<120> V197 DNA AND POLYPEPTIDES
<130> 03260.0043-00304
<140>
<141>
<150> 60/068.725
<151> 1997-12-24
<160> 2
<170> PatentIn Ver. 2.0
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<212> DNA
<213> Homo sapiens
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ggtgccttcg
ttcgccctct gtgcctcagt cctctccagtacctcaccttcatcctgcaa180
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<210> 2
<211> 153
<212> PRT
<213> Homo Sapiens
<400> 2
Met Asp Arg Leu Lys Gln Ala Arg Thr Gly
Lys Tyr Gln Lys Glu Lys
1 5 10 15
Ser Ser Thr Leu Ala Val Ala Leu Cys Ser Thr Val Pro Ser Leu Ala
20 25 30
Leu Thr Ser Leu Leu Cys Leu Gly Phe Ala Leu Gys Ala Ser Val Pro
35 40 45
Ile Leu Pro Leu Gln Tyr Leu Thr Phe Ile Leu Gln Val Ile Ser Arg
SO 50 55 60
Ser Phe Leu Tyr Gly Ser Asn Ala Ala Phe Leu Thr Leu Ala Phe Pro
65 70 75 80
Ser Glu His Phe Gly Lys Leu Phe Gly Leu Val Met Ala Leu Ser Ala
85 90 95
CA 02316016 2000-06-21
WO 99/33984 PCT/US98/27627
2
Val Val Ser Leu Leu Gln Phe Pro Ile Phe Thr Leu Ile Lys Gly Ser
100 105 110
Leu Gln Asn Asp Pro Phe Tyr Val Asn Val Met Phe Met Leu Ala Ile
S 115 120 125
Leu Leu Thr Phe Phe His Pro Phe Leu Val Tyr Arg Glu Cys Arg Thr
130 135 140
Trp Lys Glu Ser Pro Ser Ala Ile Ala
145 150
