U.S. patent application number 10/762759 was filed with the patent office on 2005-03-10 for novel human kinase protein and polynucleotides encoding the same.
Invention is credited to Abuin, Alejandro, Friedrich, Glenn, Mathur, Brian, Sands, Arthur T., Turner, C. Alexander JR., Zambrowicz, Brian.
Application Number | 20050054844 10/762759 |
Document ID | / |
Family ID | 32044896 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054844 |
Kind Code |
A1 |
Mathur, Brian ; et
al. |
March 10, 2005 |
Novel human kinase protein and polynucleotides encoding the
same
Abstract
Novel human polynucleotide and polypeptide sequences are
disclosed that can be used in therapeutic, diagnostic, and
pharmacogenomic applications.
Inventors: |
Mathur, Brian; (The
Woodlands, TX) ; Turner, C. Alexander JR.; (The
Woodlands, TX) ; Abuin, Alejandro; (The Woodlands,
TX) ; Friedrich, Glenn; (Houston, TX) ;
Zambrowicz, Brian; (The Woodlands, TX) ; Sands,
Arthur T.; (The Woodlands, TX) |
Correspondence
Address: |
Lance K. Ishimoto
LEXICON GENETICS INCORPORATED
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
32044896 |
Appl. No.: |
10/762759 |
Filed: |
January 22, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10762759 |
Jan 22, 2004 |
|
|
|
09707121 |
Nov 6, 2000 |
|
|
|
6720173 |
|
|
|
|
60164289 |
Nov 8, 1999 |
|
|
|
Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
C12N 9/1205
20130101 |
Class at
Publication: |
536/023.5 |
International
Class: |
C07H 021/04 |
Claims
1-3. Cancelled.
4. A gene delivery system comprising an isolated nucleic acid
molecule comprising the nucleic acid sequence of SEQ ID NOS: 1.
5. The gene delivery system of claim 4, wherein said isolated
nucleic acid molecule is present in a viral vector.
6. The gene delivery system of claim 4, wherein said isolated
nucleic acid molecule is present in a cationic lipid complex.
7. An isolated polypeptide comprising an amino acid sequence of SEQ
ID NO: 2.
8. An antibody having immunospecificity for a polypeptide sequence
of SEQ ID NO: 2.
Description
1. INTRODUCTION
[0001] The present application is a continuation of co-pending U.S.
application Ser. No. 09/707,121, filed on Nov. 6, 2000, which
claims the benefit of U.S. Provisional Application No. 60/164,289
which was filed on Nov. 8, 1999 and is herein incorporated by
reference in its entirety.
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding a protein that shares sequence similarity with animal
kinases. The invention encompasses the described polynucleotides,
host cell expression systems, the encoded proteins, fusion
proteins, polypeptides and peptides, antibodies to the encoded
proteins and peptides, and genetically engineered animals that
either lack or over express the disclosed genes, antagonists and
agonists of the proteins, and other compounds that modulate the
expression or activity of the proteins encoded by the disclosed
genes that can be used for diagnosis, drug screening, clinical
trial monitoring and the treatment of physiological disorders.
2. BACKGROUND OF THE INVENTION
[0003] Kinases mediate phosphorylation of a wide variety of
proteins and compounds in the cell. Along with phosphatases,
kinases are involved in a range of regulatory pathways. Given the
physiological importance of kinases, they have been subject to
intense scrutiny and are proven drug targets.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that encode a
novel human protein, and the corresponding amino acid sequences of
this protein. The novel human protein (NHP) described for the first
time herein shares structural similarity with animal kinases,
including, but not limited to serine/threonine protein kinases. As
such, the novel polynucleotides encode a new kinase protein having
homologues and orthologs across a range of phyla and species.
[0005] The novel human polynucleotides described herein, encode an
open reading frame (ORF) encoding a protein of 893 amino acids in
length (see SEQ ID NO: 2).
[0006] The invention also encompasses agonists and antagonists of
the described NHP, including small molecules, large molecules,
mutant NHPs, or portions thereof that compete with native NHP,
peptides, and antibodies, as well as nucleotide sequences that can
be used to inhibit the expression of the described NHP (e.g.,
antisense and ribozyme molecules, and gene or regulatory sequence
replacement constructs) or to enhance the expression of the
described NHP polynucleotides (e.g., expression constructs that
place the described gene under the control of a strong promoter
system). The present invention also includes both transgenic
animals that express a NHP transgene, and NHP "knock-outs" (which
can be conditional) that do not express a functional NHP.
[0007] Further, the present invention also relates to processes for
identifying compounds that modulate, i.e., act as agonists or
antagonists, of NHP expression and/or NHP product activity that
utilize purified preparations of the described NHPs and/or NHP
product, or cells expressing the same. Such compounds can be used
as therapeutic agents for the treatment of any of a wide variety of
symptoms associated with biological disorders or imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0008] The Sequence Listing provides the sequence of a novel human
ORF that encodes the described novel human kinase-like protein.
5. DETAILED DESCRIPTION OF THE INVENTION
[0009] The NHP, described for the first time herein, is a novel
protein that is expressed in, inter alia, human cell lines, and
human brain, pituitary, cerebellum, spinal cord, thymus, lymph
node, bone marrow, trachea, kidney, liver, prostate, testis,
thyroid, adrenal gland, pancreas, stomach, small intestine, colon,
skeletal muscle, uterus, placenta, mammary gland, adipose,
esophagus, bladder, cervix, rectum, pericardium, hypothalamus,
ovary, fetal kidney, and fetal lung cells. The described sequences
were compiled from gene trapped cDNAs, ESTs, a and human brain cDNA
library, (Edge Biosystems, Gaithersburg, Md.).
[0010] The present invention encompasses the nucleotides presented
in the Sequence Listing, host cells expressing such nucleotides,
the expression products of such nucleotides, and: (a) nucleotides
that encode mammalian homologs of the described genes, including
the specifically described NHP, and the NHP products; (b)
nucleotides that encode one or more portions of the NHP that
correspond to functional domains, and the polypeptide products
specified by such nucleotide sequences, including but not limited
to the novel regions of any active domain(s); (c) isolated
nucleotides that encode mutant versions, engineered or naturally
occurring, of the described NHP in which all or a part of at least
one domain is deleted or altered, and the polypeptide products
specified by such nucleotide sequences, including but not limited
to soluble proteins and peptides in which all or a portion of the
signal sequence in deleted; (d) nucleotides that encode chimeric
fusion proteins containing all or a portion of a coding region of a
NHP, or one of its domains (e.g., a receptor/ligand binding domain,
accessory protein/self-association domain, etc.) fused to another
peptide or polypeptide; or (e) therapeutic or diagnostic
derivatives of the described polynucleotides such as
oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or
gene therapy constructs comprising a sequence first disclosed in
the Sequence Listing. As discussed above, the present invention
includes: (a) the human DNA sequences presented in the Sequence
Listing (and vectors comprising the same) and additionally
contemplates any nucleotide sequence encoding a contiguous NHP open
reading frame (ORF) that hybridizes to a complement of a DNA
sequence presented in the Sequence Listing under highly stringent
conditions, e.g., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc., and
John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a
functionally equivalent gene product. Additionally contemplated are
any nucleotide sequences that hybridize to the complement of the
DNA sequence that encode and express an amino acid sequence
presented in the Sequence Listing under moderately stringent
conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree.
C. (Ausubel et al., 1989, supra), yet still encode a functionally
equivalent NHP product. Functional equivalents of a NHP include
naturally occurring NHPs present in other species and mutant NHPs
whether naturally occurring or engineered (by site directed
mutagenesis, gene shuffling, directed evolution as described in,
for example, U.S. Pat. No. 5,837,458). The invention also includes
degenerate nucleic acid variants of the disclosed NHP
polynucleotide sequences.
[0011] Additionally contemplated are polynucleotides encoding NHP
ORFs, or their functional equivalents, encoded by polynucleotide
sequences that are about 99, 95, 90, or about 85 percent similar to
corresponding regions of SEQ ID NO:1 (as measured by BLAST sequence
comparison analysis using, for example, the GCG sequence analysis
package using default parameters).
[0012] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP encoding polynucleotides. Such
hybridization conditions can be highly stringent or less highly
stringent, as described above. In instances where the nucleic acid
molecules are deoxyoligonucleotides ("DNA oligos"), such molecules
are generally about 16 to about 100 bases long, or about 20 to
about 80, or about 34 to about 45 bases long, or any variation or
combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the Sequence
Listing. Such oligonucleotides can be used in conjunction with the
polymerase chain reaction (PCR) to screen libraries, isolate
clones, and prepare cloning and sequencing templates, etc.
[0013] Alternatively, such NHP oligonucleotides can be used as
hybridization probes for screening libraries, and assessing gene
expression patterns (particularly using a micro array or
high-throughput "chip" format). Additionally, a series of the
described NHP oligonucleotide sequences, or the complements
thereof, can be used to represent all or a portion of the described
NHP sequences. The oligonucleotides, typically between about 16 to
about 40 (or any whole number within the stated range) nucleotides
in length may partially overlap each other and/or the NHP sequence
may be represented using oligonucleotides that do not overlap.
Accordingly, the described NHP polynucleotide sequence shall
typically comprise at least about two or three distinct
oligonucleotide sequences of at least about 18, and preferably
about 25, nucleotides in length that are each first disclosed in
the described Sequence Listing. Such oligonucleotide sequences may
begin at any nucleotide present within a sequence in the Sequence
Listing and proceed in either a sense (5'-to-3') orientation
vis-a-vis the described sequence or in an antisense
orientation.
[0014] For oligonucleotide probes, highly stringent conditions may
refer, e.g., to washing in 6.times.SSC/0.05% sodium pyrophosphate
at 37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NHP gene antisense molecules, useful, for example, in NHP gene
regulation (for and/or as antisense primers in amplification
reactions of NHP gene nucleic acid sequences). With respect to NHP
gene regulation, such techniques can be used to regulate biological
functions. Further, such sequences can be used as part of ribozyme
and/or triple helix sequences that are also useful for NHP gene
regulation.
[0015] Inhibitory antisense or double stranded oligonucleotides can
additionally comprise at least one modified base moiety which is
selected from the group including but not limited to
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopente- nyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0016] The antisense oligonucleotide can also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0017] In yet another embodiment, the antisense oligonucleotide
will comprise at least one modified phosphate backbone selected
from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0018] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be
used to disrupt the expression and function of a targeted NHP.
[0019] Oligonucleotides of the invention can be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides can be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), and methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0020] Low stringency conditions are well known to those of skill
in the art, and will vary predictably depending on the specific
organisms from which the library and the labeled sequences are
derived. For guidance regarding such conditions see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and
periodic updates thereof), Cold Springs Harbor Press, N.Y.; and
Ausubel et al., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y.
[0021] Alternatively, suitably labeled NHP nucleotide probes can be
used to screen a human genomic library using appropriately
stringent conditions or by PCR. The identification and
characterization of human genomic clones is helpful for identifying
polymorphisms (including, but not limited to, nucleotide repeats,
microsatellite alleles, single nucleotide polymorphisms, or coding
single nucleotide polymorphisms), determining the genomic structure
of a given locus/allele, and designing diagnostic tests. For
example, sequences derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers for use
in amplification assays to detect mutations within the exons,
introns, splice sites (e.g., splice acceptor and/or donor sites),
etc., that can be used in diagnostics and pharmacogenomics.
[0022] Further, a NHP gene homolog can be isolated from nucleic
acid from an organism of interest by performing PCR using two
degenerate or "wobble" oligonucleotide primer pools designed on the
basis of amino acid sequences within the NHP products disclosed
herein. The template for the reaction may be total RNA, mRNA,
and/or cDNA obtained by reverse transcription of mRNA prepared
from, for example, human or non-human cell lines or tissue, such as
prostate, rectum, colon, or adrenal gland, known or suspected to
express an allele of a NHP gene. The PCR product can be subcloned
and sequenced to ensure that the amplified sequences represent the
sequence of the desired NHP gene. The PCR fragment can then be used
to isolate a full length cDNA clone by a variety of methods. For
example, the amplified fragment can be labeled and used to screen a
cDNA library, such as a bacteriophage cDNA library. Alternatively,
the labeled fragment can be used to isolate genomic clones via the
screening of a genomic library.
[0023] PCR technology can also be used to isolate full length cDNA
sequences. For example, RNA can be isolated, following standard
procedures, from an appropriate cellular or tissue source (i.e.,
one known, or suspected, to express a NHP gene, such as, for
example, testis tissue). A reverse transcription (RT) reaction can
be performed on the RNA using an oligonucleotide primer specific
for the most 5' end of the amplified fragment for the priming of
first strand synthesis. The resulting RNA/DNA hybrid may then be
"tailed" using a standard terminal transferase reaction, the hybrid
may be digested with RNase H, and second strand synthesis may then
be primed with a complementary primer. Thus, cDNA sequences
upstream of the amplified fragment can be isolated. For a review of
cloning strategies that can be used, see e.g., Sambrook et al.,
1989, supra.
[0024] A cDNA encoding a mutant NHP gene can be isolated, for
example, by using PCR. In this case, the first cDNA strand may be
synthesized by hybridizing an oligo-dT oligonucleotide to mRNA
isolated from tissue known or suspected to be expressed in an
individual putatively carrying a mutant NHP allele, and by
extending the new strand with reverse transcriptase. The second
strand of the cDNA is then synthesized using an oligonucleotide
that hybridizes specifically to the 5' end of the normal gene.
Using these two primers, the product is then amplified via PCR,
optionally cloned into a suitable vector, and subjected to DNA
sequence analysis through methods well known to those of skill in
the art. By comparing the DNA sequence of the mutant NHP allele to
that of a corresponding normal NHP allele, the mutation(s)
responsible for the loss or alteration of function of the mutant
NHP gene product can be ascertained.
[0025] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry a
mutant NHP allele (e.g., a person manifesting a NHP-associated
phenotype such as, for example, immune disorders, obesity, high
blood pressure, etc.), or a cDNA library can be constructed using.
RNA from a tissue known, or suspected, to express a mutant NHP
allele. A normal NHP gene, or any suitable fragment thereof, can
then be labeled and used as a probe to identify the corresponding
mutant NHP allele in such libraries. Clones containing mutant NHP
gene sequences can then be purified and subjected to sequence
analysis according to methods well known to those skilled in the
art.
[0026] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a mutant NHP allele in an
individual suspected of or known to carry such a mutant allele. In
this manner, gene products made by the putatively mutant tissue may
be expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against a normal
NHP product, as described below. (For screening techniques, see,
for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A
Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.)
Additionally, screening can be accomplished by screening with
labeled NHP fusion proteins, such as, for example, alkaline
phosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In
cases where a NHP mutation results in an expressed gene product
with altered function (e.g., as a result of a missense or a
frameshift mutation), polyclonal antibodies to a NHP are likely to
cross-react with a corresponding mutant NHP gene product. Library
clones detected via their reaction with such labeled antibodies can
be purified and subjected to sequence analysis according to methods
well known in the art.
[0027] An additional application of the described novel human
polynucleotide sequences is their use in the molecular
mutagenesis/evolution of proteins that are at least partially
encoded by the described novel sequences using, for example,
polynucleotide shuffling or related methodologies. Such approaches
are described in U.S. Pat. Nos. 5,830,721 and 5,837,458 which are
herein incorporated by reference in their entirety.
[0028] The invention also encompasses (a) DNA vectors that contain
any of the foregoing NHP coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing NHP coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences (for example, baculo virus as described in U.S. Pat. No.
5,869,336 herein incorporated by reference); (c) genetically
engineered host cells that contain any of the foregoing NHP coding
sequences operatively associated with a regulatory element that
directs the expression of the coding sequences in the host cell;
and (d) genetically engineered host cells that express an
endogenous NHP gene under the control of an exogenously introduced
regulatory element (i.e., gene activation). As used herein,
regulatory elements include, but are not limited to, inducible and
non-inducible promoters, enhancers, operators and other elements
known to those skilled in the art that drive and regulate
expression. Such regulatory elements include but are not limited to
the human cytomegalovirus (hCMV) immediate early gene, regulatable,
viral elements (particularly retroviral LTR promoters), the early
or late promoters of SV40 adenovirus, the lac system, the trp
system, the TAC system, the TRC system, the major operator and
promoter regions of phage lambda, the control regions of fd coat
protein, the promoter for 3-phosphoglycerate kinase (PGK), the
promoters of acid phosphatase, and the promoters of the yeast
.alpha.-mating factors.
[0029] Where, as in the present instance, some of the described NHP
peptides or polypeptides are thought to be cytoplasmic proteins,
expression systems can be engineered that produce soluble
derivatives of a NHP (corresponding to a NHP extracellular and/or
intracellular domains, or truncated polypeptides lacking one or
more hydrophobic domains) and/or NHP fusion protein products
(especially NHP-Ig fusion proteins, i.e., fusions of a NHP domain
to an IgFc), NHP antibodies, and anti-idiotypic antibodies
(including Fab fragments) that can be used in therapeutic
applications. Preferably, the above expression systems are
engineered to allow the desired peptide or polypeptide to be
recovered from the culture media.
[0030] Also encompassed by the present invention are novel protein
constructs engineered in such a way that they facilitate transport
of the NHP to the target site, to the desired organ, across the
cell membrane and/or to the nucleus where the NHP can exert its
function activity. This goal can be achieved by coupling of the NHP
to a cytokine or other ligand that would direct the NHP to the
target organ and facilitate receptor mediated transport across the
membrane into the cytosol. Conjugation of NHPs to antibody
molecules or their Fab fragments could be used to target cells
bearing a particular epitope. Attaching the appropriate signal
sequence to the NHP would also transport the NHP to the desired
location within the cell. Alternatively targeting of NHP or its
nucleic acid sequence can be achieved using liposome or lipid
complex based delivery systems. Such technologies are described in
U.S. Pat. Nos. 4,594,595, 5,459,127, 5,948,767 and 6,110,490 and
their respective disclosures which are herein incorporated by
reference in their entirety.
[0031] The present invention also encompasses antibodies and
anti-idiotypic antibodies (including Fab fragments), antagonists
and agonists of the NHP, as well as compounds or nucleotide
constructs that inhibit expression of a NHP gene (transcription
factor inhibitors, antisense and ribozyme molecules, or gene or
regulatory sequence replacement constructs), or promote the
expression of a NHP (e.g., expression constructs in which NHP
coding sequences are operatively associated with expression control
elements such as promoters, promoter/enhancers, etc.).
[0032] The NHPs or NHP peptides, NHP fusion proteins, NHP
nucleotide sequences, antibodies, antagonists and agonists can be
useful for the detection of mutant NHPs or inappropriately
expressed. NHPs for the diagnosis of disease. The NHP proteins or
peptides, NHP fusion proteins, NHP nucleotide sequences, host cell
expression systems, antibodies, antagonists, agonists and
genetically engineered cells and animals can be used for screening
for drugs (or high throughput screening of combinatorial libraries)
effective in the treatment of the symptomatic or phenotypic
manifestations of perturbing the normal function of NHP in the
body. The use of engineered host cells and/or animals can offer an
advantage in that such systems allow not only for the
identification of compounds that bind to the endogenous
receptor/ligand of a NHP, but can also identify compounds that
trigger NHP-mediated activities or pathways.
[0033] Finally, the NHP products can be used as therapeutics. For
example, soluble derivatives such as NHP peptides/domains
corresponding the NHPs, NHP fusion protein products (especially
NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a
NHP, to an IgFc), NHP antibodies and anti-idiotypic antibodies
(including Fab fragments), antagonists or agonists (including
compounds that modulate or act on downstream targets in a
NHP-mediated pathway) can be used to directly treat diseases or
disorders. For instance, the administration of an effective amount
of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic
antibody (or its Fab) that mimics the NHP could activate or
effectively antagonize the endogenous NHP or a protein interactive
therewith. Nucleotide constructs encoding such NHP products can be
used to genetically engineer host cells to express such products in
vivo; these genetically engineered cells function as "bioreactors"
in the body delivering a continuous supply of a NHP, a NHP peptide,
or a NHP fusion protein to the body. Nucleotide constructs encoding
functional NHPs, mutant NHPS, as well as antisense and ribozyme
molecules can also be used in "gene therapy" approaches for the
modulation of NHP expression. Thus, the invention also encompasses
pharmaceutical formulations and methods for treating biological
disorders.
[0034] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 The NHP Sequences
[0035] The cDNA sequence and the corresponding deduced amino acid
sequence of the described NHP are presented in the Sequence
Listing. The NHP nucleotide sequences were obtained from a human
cDNA library using probes and/or primers generated from human gene
trapped sequence tags.
[0036] Expression analysis has provided evidence that the described
NHPs can be expressed in human tissues as well as gene trapped
human cells. In addition to the serine/threonine kinases, the
described NHPs also share significant similarity to a range of
additional kinase families such as cell division protein kinases,
cyclin dependent kinase, etc. from a range of phyla and species.
Given the physiological importance of protein kinases, they have
been subject to intense scrutiny as exemplified and discussed in
U.S. Pat. No. 5,817,479 herein incorporated by reference in its
entirety.
5.2 NHPs and NHP Polypeptides
[0037] NHPs, polypeptides, peptide fragments, mutated, truncated,
or deleted forms of the NHPs, and/or NHP fusion proteins can be
prepared for a variety of uses. These uses include, but are not
limited to, the generation of antibodies, as reagents in diagnostic
assays, for the identification of other cellular gene products
related to a NHP, as reagents in assays for screening for compounds
that can be as pharmaceutical reagents useful in the therapeutic
treatment of mental, biological, or medical disorders and
disease.
[0038] The Sequence Listing discloses the amino acid sequence
encoded by the described NHP-encoding polynucleotides. The NHP has
an initiator methionine in a DNA sequence context consistent with
eucaryotic translation initiation site.
[0039] The NHP amino acid sequence of the invention include the
amino acid sequence presented in the Sequence Listing as well as
analogues and derivatives thereof. Further, corresponding NHP
homologues from other species are encompassed by the invention. In
fact, any NHP protein encoded by the NHP nucleotide sequences
described above are within the scope of the invention, as are any
novel polynucleotide sequences encoding all or any novel portion of
an amino acid sequence presented in the Sequence Listing. The
degenerate nature of the genetic code is well known, and,
accordingly, each amino acid presented in the Sequence Listing, is
generically representative of the well known nucleic acid "triplet"
codon, or in many cases codons, that can encode the amino acid. As
such, as contemplated herein, the amino acid sequences presented in
the Sequence Listing, when taken together with the genetic code
(see, for example, Table 4-1 at page 109 of "Molecular Cell
Biology", 1986, J. Darnell et al. eds., Scientific American Books,
New York, N.Y., herein incorporated by reference) are generically
representative of all the various permutations and combinations of
nucleic acid sequences that can encode such amino acid
sequences.
[0040] The invention also encompasses proteins that are
functionally equivalent to the NHPs encoded by the presently
described nucleotide sequences as judged by any of a number of
criteria, including, but not limited to, the ability to bind and
modify a NHP substrate, or the ability to effect an identical or
complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, tyrosine
phosphorylation, etc.). Such functionally equivalent NHP proteins
include, but are not limited to, additions or substitutions of
amino acid residues within the amino acid sequence encoded by the
NHP nucleotide sequences described above, but which result in a
silent change, thus producing a functionally equivalent gene
product. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; positively charged (basic) amino acids include arginine,
lysine, and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid.
[0041] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. Where the
NHP peptide or polypeptide can exist, or has been engineered to
exist, as a soluble or secreted molecule, the soluble NHP peptide
or polypeptide can be recovered from the culture media. Such
expression systems also encompass engineered host cells that
express a NHP, or functional equivalent, in situ. Purification or
enrichment of a NHP from such expression systems can be
accomplished using appropriate detergents and lipid micelles and
methods well known to those skilled in the art. However, such
engineered host cells themselves may be used in situations where it
is important not only to retain the structural and functional
characteristics of the NHP, but to assess biological activity,
e.g., in drug screening assays.
[0042] The expression systems that may be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing NHP nucleotide sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing NHP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing NHP sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing NHP nucleotide sequences; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0043] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the NHP
product being expressed. For example, when a large quantity of such
a protein is to be produced for the generation of pharmaceutical
compositions of or containing NHP, or for raising antibodies to a
NHP, vectors that direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP
coding sequence may be ligated individually into the vector in
frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The PGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0044] In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. A NHP
encoding polynucleotide sequence can be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of NHP gene coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed (e.g., see Smith et
al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
[0045] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the NHP nucleotide sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing a NHP
product in infected hosts (e.g., See Logan & Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may
also be required for efficient translation of inserted NHP
nucleotide sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire NHP gene or
cDNA, including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of a NHP coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (See Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0046] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.
[0047] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the NHP sequences described above can be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the NHP product.
[0048] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci.
USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre, et al., 1984,
Gene 30:147).
[0049] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-8976). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
5.3 Antibodies to NHP Products
[0050] Antibodies that specifically recognize one or more epitopes
of a NHP, or epitopes of conserved variants of a NHP, or peptide
fragments of a NHP are also encompassed by the invention. Such
antibodies include but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0051] The antibodies of the invention can be used, for example, in
the detection of NHP in a biological sample and may, therefore, be
utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of NHP. Such antibodies
may also be utilized in conjunction with, for example, compound
screening schemes for the evaluation of the effect of test
compounds on expression and/or activity of a NHP gene product.
Additionally, such antibodies can be used in conjunction gene
therapy to, for example, evaluate the normal and/or engineered
NHP-expressing cells prior to their introduction into the patient.
Such antibodies may additionally be used as a method for the
inhibition of abnormal NHP activity. Thus, such antibodies may,
therefore, be utilized as part of treatment methods.
[0052] For the production of antibodies, various host animals may
be immunized by injection with the NHP, an NHP peptide (e.g., one
corresponding the a functional domain of an NHP), truncated NHP
polypeptides (NHP in which one or more domains have been deleted),
functional equivalents of the NHP or mutated variant of the NHP.
Such host animals may include but are not limited to pigs, rabbits,
mice, goats, and rats, to name but a few. Various adjuvants may be
used to increase the immunological response, depending on the host
species, including but not limited to Freund's adjuvant (complete
and incomplete), mineral salts such as aluminum hydroxide or
aluminum phosphate, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Alternatively, the
immune response could be enhanced by combination and or coupling
with molecules such as keyhole limpet hemocyanin, tetanus toxoid,
diptheria toxoid, ovalbumin, cholera toxin or fragments thereof.
Polyclonal antibodies are heterogeneous populations of antibody
molecules derived from the sera of the immunized animals.
[0053] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0054] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region. Such
technologies are described in U.S. Pat. Nos. 6,075,181 and
5,877,397 and their respective disclosures which are herein
incorporated by reference in their entirety.
[0055] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be
adapted to produce single chain antibodies against NHP gene
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0056] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0057] Antibodies to a NHP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NHP, using techniques
well known to those skilled in the art. (See, e.g., Greenspan &
Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438). For example antibodies which bind to a NHP
domain and competitively inhibit the binding of NHP to its cognate
receptor/ligand can be used to generate anti-idiotypes that "mimic"
the NHP and, therefore, bind, activate, or neutralize a NHP, NHP
receptor, or NHP ligand. Such anti-idiotypic antibodies or Fab
fragments of such anti-idiotypes can be used in therapeutic
regimens involving a NHP mediated pathway.
[0058] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. All cited publications, patents, and
patent applications are herein incorporated by reference in their
entirety.
Sequence CWU 1
1
2 1 2682 DNA homo sapiens 1 atgtttcccc tgaaggacgc tgaaatggga
gcctttacct tctttgcctc ggctctgcca 60 catgatgttt gtggaagcaa
tggacttcct ctcacaccaa attccatcaa aattttaggg 120 cgctttcaaa
tccttaaaac catcacccat cccagactct gccagtatgt ggatatttct 180
aggggaaagc atgaacgact agtggtcgtg gctgaacatt gtgaacgtag tctggaagac
240 ttgcttcgag aaaggaaacc tgtgagctgt tcaacggttt tgtgtatagc
atttgaggtt 300 cttcagggct tgcagtatat gaacaaacat ggtatagtac
acagggcatt gtctcctcat 360 aatatcctgt tggaccgaaa gggacatatt
aaattggcta aatttggact ttatcacatg 420 acagctcatg gtgatgatgt
tgatttccca atagggtatc cctcgtactt ggcccctgag 480 gtaattgcac
agggaatttt caaaaccact gatcacatgc caagtaaaaa accattgcct 540
tctggcccca aatcagatgt atggtctctt ggaatcattt tatttgagct ttgtgtggga
600 agaaaattat ttcagagctt ggatatttct gaaagactaa aatttttgct
tactttggat 660 tgtgtagatg acactttaat agttctggct gaagagcatg
gttgtttgga cattataaag 720 gagcttcctg aaactgtgat agatcttttg
aataagtgcc ttaccttcca tccttctaag 780 aggccaaccc cagatgaatt
aatgaaggac aaagtattca gtgaggtatc acctttatat 840 acccccttta
ccaaacctgc cagtctgttt tcatcttctc tgagatgtgc tgatttaact 900
ctgcctgagg atatcagtca gttgtgtaaa gatataaata atgattacct ggcagaaaga
960 tctattgaag aagtgtatta cctttggtgt ttggctggag gtgacttgga
gaaagagctt 1020 gtcaacaagg aaatcattcg atccaaacca cctatctgca
cactccccaa ttttctcttt 1080 gaggatggtg aaagctttgg acaaggtcga
gatagaagct cgcttttaga tgataccact 1140 gtgacattgt cgttatgcca
gctaagaaat agattgaaag atgttggtgg agaagcattt 1200 tacccattac
ttgaagatga ccagtctaat ttacctcatt caaacagcaa taatgagttg 1260
tctgcagctg ccacgctccc tttaatcatc agagagaagg atacagagta ccaactaaat
1320 agaattattc tcttcgacag gctgctaaag gcttatccat ataaaaaaaa
ccaaatctgg 1380 aaagaagcaa gagttgacat tcctcctctt atgagaggtt
taacctgggc tgctcttctg 1440 ggagttgagg gagctattca tgccaagtac
gatgcaattg ataaagacac tccaattcct 1500 acagatagac aaattgaagt
ggatattcct cgctgtcatc agtacgatga actgttatca 1560 tcaccagaag
gtcatgcaaa atttaggcgt gtattaaaag cctgggtagt gtctcatcct 1620
gatcttgtgt attggcaagg tcttgactca ctttgtgctc cattcctata tctaaacttc
1680 aataatgaag ccttggctta tgcatgtatg tctgctttta ttcccaaata
cctgtataac 1740 ttcttcttaa aagacaactc acatgtaata caagagtatc
tgactgtctt ctctcagatg 1800 attgcatttc atgatccaga gctgagtaat
catctcaatg agattggttt cattccagat 1860 ctctatgcca tcccttggtt
tcttaccatg tttactcatg tatttccact acacaaaatt 1920 ttccacctct
gggatacctt actacttggg aattcctctt tcccattctg tattggagta 1980
gcaattcttc agcagctgcg ggaccggctt ttggctaatg gctttaatga gtgtattctt
2040 ctcttctccg atttaccaga aattgacatt gaacgctgtg tgagagaatc
tatcaacctg 2100 ttttgttgga ctcctaaaag tgctacttac agacagcatg
ctcaacctcc aaagccatct 2160 tctgacagca gtggaggcag aagttcggca
ccttatttct ctgctgagtg tccagatcct 2220 ccaaagacag atctgtcaag
agaatccatc ccattaaatg acctgaagtc agaagtatca 2280 ccacggattt
cagcagagga cctgattgac ttgtgtgagc tcacagtgac aggccacttc 2340
aaaacaccca gcaagaaaac aaagtccagt aaaccaaagc tcctggtggt tgacatccgg
2400 aatagtgaag actttattcg tggtcacatt tcaggaagca tcaacattcc
attcagtgct 2460 gccttcactg cagaagggga gcttacccag ggcccttaca
ctgctatgct ccagaacttc 2520 aaagggaagg tcattgtcat cgtggggcat
gtggcaaaac acacagctga gtttgcagct 2580 caccttgtga agatgaaata
tccaagaatc tgtattctag atggtggcat taataaaata 2640 aagccaacag
gcctcctcac catcccatct cctcaaatat ga 2682 2 893 PRT homo sapiens 2
Met Phe Pro Leu Lys Asp Ala Glu Met Gly Ala Phe Thr Phe Phe Ala 1 5
10 15 Ser Ala Leu Pro His Asp Val Cys Gly Ser Asn Gly Leu Pro Leu
Thr 20 25 30 Pro Asn Ser Ile Lys Ile Leu Gly Arg Phe Gln Ile Leu
Lys Thr Ile 35 40 45 Thr His Pro Arg Leu Cys Gln Tyr Val Asp Ile
Ser Arg Gly Lys His 50 55 60 Glu Arg Leu Val Val Val Ala Glu His
Cys Glu Arg Ser Leu Glu Asp 65 70 75 80 Leu Leu Arg Glu Arg Lys Pro
Val Ser Cys Ser Thr Val Leu Cys Ile 85 90 95 Ala Phe Glu Val Leu
Gln Gly Leu Gln Tyr Met Asn Lys His Gly Ile 100 105 110 Val His Arg
Ala Leu Ser Pro His Asn Ile Leu Leu Asp Arg Lys Gly 115 120 125 His
Ile Lys Leu Ala Lys Phe Gly Leu Tyr His Met Thr Ala His Gly 130 135
140 Asp Asp Val Asp Phe Pro Ile Gly Tyr Pro Ser Tyr Leu Ala Pro Glu
145 150 155 160 Val Ile Ala Gln Gly Ile Phe Lys Thr Thr Asp His Met
Pro Ser Lys 165 170 175 Lys Pro Leu Pro Ser Gly Pro Lys Ser Asp Val
Trp Ser Leu Gly Ile 180 185 190 Ile Leu Phe Glu Leu Cys Val Gly Arg
Lys Leu Phe Gln Ser Leu Asp 195 200 205 Ile Ser Glu Arg Leu Lys Phe
Leu Leu Thr Leu Asp Cys Val Asp Asp 210 215 220 Thr Leu Ile Val Leu
Ala Glu Glu His Gly Cys Leu Asp Ile Ile Lys 225 230 235 240 Glu Leu
Pro Glu Thr Val Ile Asp Leu Leu Asn Lys Cys Leu Thr Phe 245 250 255
His Pro Ser Lys Arg Pro Thr Pro Asp Glu Leu Met Lys Asp Lys Val 260
265 270 Phe Ser Glu Val Ser Pro Leu Tyr Thr Pro Phe Thr Lys Pro Ala
Ser 275 280 285 Leu Phe Ser Ser Ser Leu Arg Cys Ala Asp Leu Thr Leu
Pro Glu Asp 290 295 300 Ile Ser Gln Leu Cys Lys Asp Ile Asn Asn Asp
Tyr Leu Ala Glu Arg 305 310 315 320 Ser Ile Glu Glu Val Tyr Tyr Leu
Trp Cys Leu Ala Gly Gly Asp Leu 325 330 335 Glu Lys Glu Leu Val Asn
Lys Glu Ile Ile Arg Ser Lys Pro Pro Ile 340 345 350 Cys Thr Leu Pro
Asn Phe Leu Phe Glu Asp Gly Glu Ser Phe Gly Gln 355 360 365 Gly Arg
Asp Arg Ser Ser Leu Leu Asp Asp Thr Thr Val Thr Leu Ser 370 375 380
Leu Cys Gln Leu Arg Asn Arg Leu Lys Asp Val Gly Gly Glu Ala Phe 385
390 395 400 Tyr Pro Leu Leu Glu Asp Asp Gln Ser Asn Leu Pro His Ser
Asn Ser 405 410 415 Asn Asn Glu Leu Ser Ala Ala Ala Thr Leu Pro Leu
Ile Ile Arg Glu 420 425 430 Lys Asp Thr Glu Tyr Gln Leu Asn Arg Ile
Ile Leu Phe Asp Arg Leu 435 440 445 Leu Lys Ala Tyr Pro Tyr Lys Lys
Asn Gln Ile Trp Lys Glu Ala Arg 450 455 460 Val Asp Ile Pro Pro Leu
Met Arg Gly Leu Thr Trp Ala Ala Leu Leu 465 470 475 480 Gly Val Glu
Gly Ala Ile His Ala Lys Tyr Asp Ala Ile Asp Lys Asp 485 490 495 Thr
Pro Ile Pro Thr Asp Arg Gln Ile Glu Val Asp Ile Pro Arg Cys 500 505
510 His Gln Tyr Asp Glu Leu Leu Ser Ser Pro Glu Gly His Ala Lys Phe
515 520 525 Arg Arg Val Leu Lys Ala Trp Val Val Ser His Pro Asp Leu
Val Tyr 530 535 540 Trp Gln Gly Leu Asp Ser Leu Cys Ala Pro Phe Leu
Tyr Leu Asn Phe 545 550 555 560 Asn Asn Glu Ala Leu Ala Tyr Ala Cys
Met Ser Ala Phe Ile Pro Lys 565 570 575 Tyr Leu Tyr Asn Phe Phe Leu
Lys Asp Asn Ser His Val Ile Gln Glu 580 585 590 Tyr Leu Thr Val Phe
Ser Gln Met Ile Ala Phe His Asp Pro Glu Leu 595 600 605 Ser Asn His
Leu Asn Glu Ile Gly Phe Ile Pro Asp Leu Tyr Ala Ile 610 615 620 Pro
Trp Phe Leu Thr Met Phe Thr His Val Phe Pro Leu His Lys Ile 625 630
635 640 Phe His Leu Trp Asp Thr Leu Leu Leu Gly Asn Ser Ser Phe Pro
Phe 645 650 655 Cys Ile Gly Val Ala Ile Leu Gln Gln Leu Arg Asp Arg
Leu Leu Ala 660 665 670 Asn Gly Phe Asn Glu Cys Ile Leu Leu Phe Ser
Asp Leu Pro Glu Ile 675 680 685 Asp Ile Glu Arg Cys Val Arg Glu Ser
Ile Asn Leu Phe Cys Trp Thr 690 695 700 Pro Lys Ser Ala Thr Tyr Arg
Gln His Ala Gln Pro Pro Lys Pro Ser 705 710 715 720 Ser Asp Ser Ser
Gly Gly Arg Ser Ser Ala Pro Tyr Phe Ser Ala Glu 725 730 735 Cys Pro
Asp Pro Pro Lys Thr Asp Leu Ser Arg Glu Ser Ile Pro Leu 740 745 750
Asn Asp Leu Lys Ser Glu Val Ser Pro Arg Ile Ser Ala Glu Asp Leu 755
760 765 Ile Asp Leu Cys Glu Leu Thr Val Thr Gly His Phe Lys Thr Pro
Ser 770 775 780 Lys Lys Thr Lys Ser Ser Lys Pro Lys Leu Leu Val Val
Asp Ile Arg 785 790 795 800 Asn Ser Glu Asp Phe Ile Arg Gly His Ile
Ser Gly Ser Ile Asn Ile 805 810 815 Pro Phe Ser Ala Ala Phe Thr Ala
Glu Gly Glu Leu Thr Gln Gly Pro 820 825 830 Tyr Thr Ala Met Leu Gln
Asn Phe Lys Gly Lys Val Ile Val Ile Val 835 840 845 Gly His Val Ala
Lys His Thr Ala Glu Phe Ala Ala His Leu Val Lys 850 855 860 Met Lys
Tyr Pro Arg Ile Cys Ile Leu Asp Gly Gly Ile Asn Lys Ile 865 870 875
880 Lys Pro Thr Gly Leu Leu Thr Ile Pro Ser Pro Gln Ile 885 890
* * * * *