Note: Descriptions are shown in the official language in which they were submitted.
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hOB-8P2h COMPOSITIONS, METHODS AND USES THEREOF
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to compounds and
compositions comprising novel human obesity protein binding
protein-2 homolog (hOB-BP2h) polypeptides, nucleic acids,
host cells, transgenics, chimerics, antibodies,
compositions, and methods of making and using thereof.
RELATED ART
Obesity, and especially upper body obesity, is a common
and very serious public health problem in the United States
and throughout the world, and is expected to worsen as the
population ages. Currently, about 33~ of Americans are
overweight enough to be unhealthy (i.e., body weight greater
than 26 percent above standard weight guidelines). The
proportion of obese adults among the well-fed populations of
the world is expected to rise to more than 50~ within 20
years.
2o Numerous studies indicate that lowering body weight
dramatically reduces risk for chronic diseases such as
diabetes, hypertension, hyperlipidemia, coronary heart
disease, cancer, and muscularoskeletal diseases. Recent
estimates for the medical cost of obesity are 150 billion
dollars ($US) world-wide. Although the precise cause of
obesity is not known, obese patients may lose weight through
deliberate modification of behavior, such as changing diet
and increasing exercise. Unfortunately, an estimated 33
billion dollars ($US) are spent each year on such weight-
loss measures that are largely futile, with failure rates
reaching 95~. Failure may be due to genetic factors that
cause increased appetite, a preference for high-fat foods,
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or a tendency for lipogenic metabolism. People inheriting
such genetic traits are prone to becoming obese regardless
of their efforts to combat the condition. Therefore, a
pharmacological agent that can correct this adiposity
handicap and allow the physician to successfully treat obese
patients in spite of their genetic inheritance is needed.
The obesity gene (Ob gene) has been demonstrated to
encode a protein, termed leptin, which appears to decrease
appetite and control energy metabolism. Studies have shown
io mice deficient in active Ob gene product or carrying an Ob
gene mutation (ob/ob) are grossly obese and develop diabetes
mellitus, while injection of leptin causes the mice to curb
their food intake and shed fat. See, e.g., Barinaga (1995)
Science 269:475-476; Zhang, et al. (1994) Nature 372:425-
432; Pelleymounter, et al. (1995) Science 269:540-543;
Halaas, et al. (1995) Science 269:543-546.
Leptin is primarily secreted by adipose tissue, and is
thought to exert its effects by interactions with specific
receptors, e.g., in the hypothalamus. In humans,
2o circulating leptin levels are increased in obesity and
regulated by fasting, feeding, and body weight changes.
Unfortunately, the obesity protein disclosed by Zhang
et al. and other obesity proteins and analogs subsequently
disclosed by others are poor pharmaceutical agents for two
reasons: they have a short half-life in the circulation
[about 25 minutes; Klein, S., et al., Diabetes 4 5:984-987
(1996)]; and they are unstable in solution formulations,
especially physically unstable.
In blood, leptin is thought to be bound to other
3o proteins. See, e.g., Houseknecht, et al. (1996) Diabetes
45:1638-1643; Sinha, et al. (1996) J. of Clin. Invest.
98:1277-1282; Diamond, et al. (1997) Biochem. Biophys. Res.
Comm. 233:818-822. Modulating binding protein systems are
known to exist for numerous circulating hormones, growth
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factors, and cytokines including steroid and thyroid
hormones, TGF(31, TGF(32, TNFOC, IGF-1, and IGF-II. See
Bonner, J.C., and Brody, A.R. (1995) Am. J. Physiol.
268:L869-L878. These binding proteins often serve to alter
the clearance rate of hormones or cytokines, increase or
decrease the biological activity of the ligand, and provide
responsiveness to previously unresponsive cells. Leptin
binding proteins in blood could similarly serve to modulate
the active form of leptin.
Generally, hormone action depends on the interaction of
soluble hormone with receptor proteins expressed by
responsive cells. The classical concepts of hormone action
considered the hormone or humoral "liquid" factor to be
mobile in body fluids and the receptors to be fixed to
cells. However, over the past several years many receptors
have been found to have soluble isoforms in addition to the
membrane-bound forms that are traditionally thought of as
the mediators of ligand-induced signal transduction.
Receptors for growth hormone, G-CSF, GM-CSF, IL-4, IL-5, IL-
6, IL-7, leukemia inhibitory factor (LIF), and ciliary
neurotropic factor (CNTF) in the hematopoietin superfamily,
as well as IL-1, IL-2, (IL-2 receptor ?), tumor necrosis
factor (TNF), M-CSF/CSF-1, EGF, and NGF have been isolated
either as soluble proteins or as alternatively processed
forms that are predicted on the basis of their cDNA sequence
to encode soluble receptors. The soluble isoforms of G-CSF,
GM-CSF, IL-4, IL-5, IL-7, and LIF receptors appear to arise
as a result of alternative mRNA splicing. The soluble IL-1,
IL-2R, and TNF receptors appear to be released from the cell
3o surface by proteolytic cleavage of the extracellular domains
of the membrane-associated receptors.
There are indications that soluble receptors have
physiologic and pathologic significance. Elevated serum
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levels of soluble TNF receptor are evident in some patients
with hairy cell leukemia and chronic lymphocytic leukemia,
and soluble IL-2R is seen in the serum and ascites of some
patients with ovarian cancer.
The physiologic roles of the soluble receptors are
incompletely understood. Some soluble receptors act as
serum binding proteins and are believed to stabilize their
ligands (hormone). They are thought to have no intrinsic
role in signal transduction and only function to prevent
1o degradation of their ligand until it is delivered to the
membrane-associated receptor. Soluble receptors can also
compete with their membrane-bound counterparts for binding
to ligand. Thus, in the presence of sufficient amounts of
soluble receptor, ligand binding to the membrane-associated
receptor is diminished and signaling is dampened or
inhibited. In this scenario, the soluble receptor
"modulates" concentrations of active ligand in the
extracellular milieu.
The action of membrane-associated ligands when
2o contrasted with that of signal-altering soluble receptors
blurs the distinction between what comprises a receptor and
what comprises a ligand. Furthermore, soluble receptors may
be generated in the laboratory even from receptors that do
not express a native soluble isoform. The construction and
development of soluble receptors as pharmaceuticals may be
useful to specifically inhibit or facilitate hormone action
in disease states.
Because of the desire to provide a more effective
pharmaceutical treatment of the severe problem of obesity,
3o there exists a need to isolate and provide leptin binding
proteins that may effectively increase the plasma half-life
of circulating obesity protein, improve the physical
stability of obesity protein formulations, or act
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independently to provide a more suitable treament for
obesity. Accordingly, the present invention provides
hOB-BP2h polypeptides, nucleic acids, host cells,
transgenics, chimerics, as well as methods of making and
using thereof.
SZJN~lARY OF THE INVENTION
The present invention provides isolated nucleic acids
and encoded hOB-BP2h polypeptides, including specified
1o fragments and variants thereof, as well as hOB-BP2h
compositions, probes, primers, vectors, host cells,
antibodies, transgenics, chimerics and methods of making and
using thereof, as described and enabled herein.
The present invention provides, in one aspect, isolated
nucleic acid molecules comprising or complementary to a
polynucleotide encoding specific hOB-BP2h polypeptides, as
well as fragments or specified variants comprising at least
one domain thereof.
Such polypeptides are provided as non-limiting examples
2o by the corresponding domains, specified fragments, and
specified variants of hOB-BP2h polypeptides corresponding to
at least 90-100 of the contiguous amino acids of at least
one of SEQ ID N0:3, 4, or 5.
The present invention further provides recombinant
vectors, comprising 1-40 of said isolated hOB-BP2h nucleic
acid molecules of the present invention, host cells
containing such nucleic acids and recombinant vectors, as
well as methods of making and using such nucleic acid,
vectors and host cells.
3o The present invention also provides methods of making
or using such nucleic acids, vectors and host cells, such as
but not limited to, using them for the production of hOB-
BP2h nucleic acids and polypeptides by known recombinant,
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synthetic and purification techniques, based on the teaching
and guidance presented herein in combination with what is
known in the art.
The present invention also provides an isolated hOB-
BP2h polypeptide, comprising at least one fragment, domain
or specified variant of at least 90-100 of the contiguous
amino acids of at least one portion of at least one of SEQ
ID N0:3, 4, or 5.
The present invention also provides an isolated hOB-
to BP2h polypeptide as described herein, wherein the
polypeptide further comprises at least one specified
substitution, insertion or deletion corresponding to
portions or residues of at least one of SEQ ID N0:3, 4, or
5.
The present invention also provides an isolated hOB-
BP2h polypeptide as described herein, wherein the
polypeptide has at least one activity, such as, but not
limited to, leptin binding, weight loss, and regulation of
adiposity (Reitman, et al. (1997) Journal of Biological
2o Chemistry, 272:(48):30546-30551; Halaas, et al. (1995)
Science, 269:540-542; Pellyemounter, et al. (1995) Science
269:540-543). A hOB-BP2h polypeptide can thus be screened
for a corresponding activity according to known methods.
The present invention also provides a composition
comprising an isolated hOB-BP2h nucleic acid and polypeptide
as described herein and a carrier or diluent. The carrier
or diluent can optionally be pharmaceutically acceptable,
according to known methods.
The present invention also provides an isolated nucleic
3o acid probe, primer or fragment, as described herein, wherein
the nucleic acid comprises a polynucleotide of at least 10
nucleotides, corresponding or complementary to at least 10
nucleotides of at least one of SEQ ID N0:1, SEQ ID N0:2, or
a consensus sequence thereof.
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The present invention also provides a recombinant
vector comprising an isolated hOB-BP2h nucleic acid as
described herein.
The present invention also provides a host cell,
comprising an isolated hOB-BP2h nucleic acid as described
herein.
The present invention also provides a method for
constructing a recombinant host cell that expresses a hOB-
BP2h polypeptide, comprising introducing into the host cell
1o a hOB-BP2h nucleic acid in replicatable form as described
herein to provide the recombinant host cell. The present
invention also provides a recombinant host cell provided by
a method as described herein.
The present invention also provides a method for
expressing at least one hOB-BP2h polypeptide in a
recombinant host cell, comprising culturing a recombinant
host cell as described herein under conditions wherein at
least one hOB-BP2h polypeptide is expressed in detectable or
recoverable amounts.
The present invention also provides an isolated hOB-
BP2h polypeptide produced by a recombinant, synthetic, and
any suitable purification method as described herein and as
known in the art.
The present invention also provides a pharmaceutical
formulation containing as an active ingredient a hOB-BP2h
polypeptide and/or nucleic acid composition as described
herein.
Furthermore, the present invention provides a method of
treating obesity or obesity related diseases by
3o administering a pharmaceutical formulation of the compounds
and/or compositions of the present invention. Such methods
of treating obesity or obesity related disorders are well
within the skill of an ordinarily skilled artisan provided
the inventions disclosed herein. An example of such
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knowledge in the art includes, but is not limited to, PCT
Publication No. 98/06752 which is entirely incorporated by
reference herein.
The present invention also provides a hOB-BP2h antibody
or fragment, comprising a polyclonal and monoclonal antibody
or fragment that specifically binds at least one epitope
specific to at least one hOB-BP2h polypeptide as described
herein.
The present invention also provides a method for
1o producing a hOB-BP2h antibody or antibody fragment,
comprising generating the antibody or fragment that binds at
least one epitope that is specific to an isolated hOB-BP2h
polypeptide as described herein, the generating done by
known recombinant, synthetic and hybridoma methods.
The present invention also provides a hOB-BP2h antibody
or fragment produced by a method as described herein or as
known in the art.
The present invention also provides a method for
identifying compounds that bind a hOB-BP2h polypeptide,
2o comprising
a) admixing at least one isolated hOB-BP2h
polypeptide as described herein with a test compound or
composition; and
b) detecting at least one binding interaction
between the polypeptide and the compound or composition,
optionally further comprising detecting a change in
biological activity, such as a reduction or increase.
DESCRIPTION OF THE INVENTION
The present invention provides isolated, recombinant
3o and synthetic nucleic acid molecules comprising at least one
polynucleotide encoding at least one hOB-BP2h polypeptide
comprising specific full length sequences, fragments and
specified variants thereof, such polypeptides, and methods
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of making and using said nucleic acids and polypeptides
thereof. A hOB-BP2h polypeptide of the invention comprises
at least one fragment, domain, and specified variant as a
portion or fragment of a hOB-BP2h protein as described
herein.
Utili ty
The present invention also provides at least one utility
by providing isolated nucleic acid comprising polynucleotides
of sufficient length and complementarity to a hOB-BP2h
1o nucleic acid for use as probes or amplification primers in
the detection, quantitation, or isolation of gene sequences
or transcripts. For example, isolated nucleic acids of the
present invention can be used as probes for detecting
deficiencies in the level of mRNA, in screens for detection
of mutations in at least one hOB-BP2h gene (e. g.,
substitutions, deletions, or additions), or for monitoring
up-regulation of expression of said gene, or changes in
biological activity as described herein in screening assays
of compounds, and for detection of any number of allelic
2o variants (polymorphisms or isoforms) of the gene.
The isolated nucleic acids of the present invention can
also be used for recombinant expression of hOB-BP2h
polypeptides, or for use as immunogens in the preparation and
screening of antibodies. The isolated nucleic acids of the
present invention can also be employed for use in sense or
antisense suppression of one or more hOB-BP2h genes or
nucleic acids, in a host cell, or tissue in vivo or in vitro.
Attachment of chemical agents which bind, intercalate,
cleave and crosslink to the isolated nucleic acids of the
3o present invention can also be used to modulate transcription
or translation of at least one nucleic acid disclosed herein.
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Citations
All publications or patents cited herein are entirely
incorporated herein by reference as they show the state of
the art at the time of the present invention to provide
description and enablement of the present invention.
Publications refer to scientific, patent publication or any
other information available in any media format, including
all recorded, electronic or printed formats. The following
citations are entirely incorporated by reference: Ausubel,
et al., eds., Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y., N.Y. (1987-1998); Coligan et al., eds.,
Current Protocols in Protein Science, John Wiley & Sons,
Inc., N.Y., N.Y. (1995-1999); Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a
Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Coligan,
et al., eds., Current Protocols in Immunology, John Wiley &
Sons, N.Y., N.Y. (1992-1999).
Definitions
2o The following definitions of terms are intended to
correspond to those as well known in the art. The following
terms are therefore not limited to the definitions given,
but are used according to the state of the art, as
demonstrated by cited and contemporary publications or
patents.
A "polynucleotide" comprises at least 10-20 nucleotides
of a nucleic acid (RNA, DNA or combination thereof), provided
by any means, such as synthetic, recombinant isolation or
purification method steps.
The terms "complementary" or "complementarity" as used
herein refer to the capacity of purine, pyrimidine,
synthetic or modified nucleotides to associate by partial or
complete complementarity through hydrogen or other bonding
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to form partial or complete double- or triple-stranded
nucleic acid molecules. The following base pairs occur by
complete complementarity: (i) guanine (G) and cytosine (C);
(ii) adenine (A) and thymine (T); and adenine (A) and uracil
(U). "Partial complementarity" refers to association of two
or more bases by one or more hydrogen bonds or attraction
that is less than the complete complementarity as described
above. Partial or complete complementarity can occur
between any two nucleotides, including naturally occurring
or modified bases, e.g., as listed in 37 CFR ~ 1.822. All
such nucleotides are included in polynucleotides of the
invention as described herein.
The term "fusion protein" denotes a hybrid protein
molecule not found in nature comprising a translational
fusion or enzymatic fusion in which two or more different
proteins or fragments thereof are covalently linked on a
single polypeptide chain. The term "polypeptide" also
includes such fusion proteins.
"Host cell" refers to any eucaryotic, procaryotic, or
2o fusion or other cell or pseudo cell or membrane-containing
construct that is suitable for propagating and expressing an
isolated nucleic acid that is introduced into a host cell by
any suitable means known in the art (e. g., but not limited
to, transformation or transfection, or the like), or induced
to express an endogenous nucleic acid encoding a hOB-BP2h
polypeptide according to the present invention. The cell can
be part of a tissue or organism, isolated in culture or in
any other suitable form.
The term "hybridization" as used herein refers to a
3o process in which a partially or completely single-stranded
nucleic acid molecule joins with a complementary strand
through nucleotide base pairing. Hybridization can occur
under conditions of low, moderate or high stringency, with
high stringency preferred. The degree of hybridization
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depends upon, for example, the degree of homology, the
stringency conditions, and the length of hybridizing strands
as known in the art.
By "isolated" nucleic acid molecules) is intended a
nucleic acid molecule, DNA, RNA, or both which has been
removed from its native or naturally occurring environment.
For example, recombinant nucleic acid molecules contained or
generated in culture, a vector and a host cell are
considered isolated for the purposes of the present
1o invention. Further examples of isolated nucleic acid
molecules include recombinant nucleic acid molecules
maintained in heterologous host cells or purified (partially
or substantially) nucleic acid molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the nucleic acid molecules of the present
invention. Isolated nucleic acid molecules according to the
present invention further include such molecules produced
synthetically, purified from or provided in cells containing
such nucleic acids, where the nucleic acid exists in other
2o than a naturally occurring form, quantitatively or
qualitatively.
"Isolated" used in reference to at least one
polypeptide of the invention describes a state of isolation
such that the peptide or polypeptide is not in a naturally
occurring form and has been purified to remove at least some
portion of cellular or non-cellular molecules with which the
protein is naturally associated. However, "isolated" may
include the addition of other functional or structural
polypeptides for a specific purpose, where the other peptide
3o may occur naturally associated with at least one polypeptide
of the present invention, but for which the resulting
compound or composition does not exist naturally.
The term "mature protein" or "mature polypeptide" as
used herein refers to the forms) of the protein produced by
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expression in a mammalian cell. It is generally
hypothesized that once export of a growing protein chain
across the rough endoplasmic reticulum has been initiated,
proteins secreted by mammalian cells have a signal peptide
(SP) sequence which is cleaved from the complete polypeptide
to produce a "mature" form of the protein. Oftentimes,
cleavage of a secreted protein is not uniform and may result
in more than one species of mature protein. The cleavage
site of a secreted protein is determined by the primary
1o amino acid sequence of the complete protein and generally
can not be predicted with complete accuracy.
Methods for predicting whether a protein has a SP
sequence, as well as the cleavage point for that sequence,
are available. The analysis of the amino acid sequence of
the proteins described herein indicated the cleavage point
is after amino acid 30-50, preferably between 40 and 41, as
presented in SEQ ID NOS:3 or 4. The resulting mature
proteins are presented as non-limiting examples. As one of
ordinary skill would appreciate, however, cleavage sites
sometimes vary from organism to organism and cannot be
predicted with absolute certainty.
Accordingly, the present invention provides
polypeptides having a sequence of 90-100 of the contiguous
sequence shown in SEQ ID N0: 3 and SEQ ID N0:4 which have an
N-terminus beginning within 10 residues (i.e., + or - 10
residues) of the predicted cleavage point of SEQ ID N0:3 or
4. However, cleavage sites for a secreted protein may be
determined experimentally by amino-terminal sequencing of
the one or more species of mature proteins found within a
3o purified preparation of the protein.
A "nucleic acid probe," "oligonucleotide probe," or
"probe" as used herein comprises at least one detestably
labeled or unlabeled nucleic acid which hybridizes under
specified hybridization conditions with at least one other
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nucleic acid. This term also refers to a single- or
partially double-stranded nucleic acid, oligonucleotide or
polynucleotide that will associate with a complementary or
partially complementary target nucleic acid to form at least
a partially double-stranded nucleic acid molecule. A
nucleic acid probe may be an oligonucleotide or a nucleotide
polymer. A probe can optionally contain a detectable moiety
which may be attached to the ends) of the probe or be
internal to the sequence of the probe, termed a "detectable
1o probe" or "detectable nucleic acid probe."
A "primer" is a nucleic acid fragment or
oligonucleotide which functions as an initiating substrate
for enzymatic or synthetic elongation of, for example, a
nucleic acid molecule, e.g., using an amplification
reaction, such as, but not limited to, a polymerase chain
reaction (PCR), as known in the art.
The term "stringency" refers to hybridization
conditions for nucleic acids in solution. High stringency
conditions disfavor non-homologous base pairing. Low
2o stringency conditions have much less of this effect.
Stringency may be altered, for example, by changes in
temperature and salt concentration, or other conditions, as
well known in the art.
A non-limiting example of "high stringency" conditions
includes, for example, (a) a temperature of about 42 °C , a
formamide concentration of about s 20~, and a low salt (SSC)
concentration, or, alternatively, a temperature of about
65°C, or less, and a low salt (SSPE) concentration; (b)
hybridization in 0.5 M NaHP04, 7$ sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65 °C (See, e.g., Ausubel, et al., ed.,
Current Protocols in Molecular Biology, 1987-1998, Wiley
Interscience, New York, at ~2.10.3). "SSC" comprises a
hybridization and wash solution. A stock 20X SSC solution
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contains 3M sodium chloride, 0.3M sodium citrate, pH 7Ø
"SSPE" comprises a hybridization and wash solution. A 1X
SSPE solution contains 180 mM NaCl, 9mM Na2HP04, 0.9 mM
NaH2P04 and 1 mM EDTA, pH 7.4.
The term "vector" as used herein refers to a nucleic
acid compound used for introducing exogenous or endogenous
nucleic acid into host cells. A vector comprises a
nucleotide sequence which may encode one or more polypeptide
molecules. Plasmids, cosmids, viruses and bacteriophages,
1o in a natural state or which have undergone recombinant
engineering, are non-limiting examples of commonly used
vectors to provide recombinant vectors comprising at least
one desired isolated nucleic acid molecule.
Nucleic Acid Molecules
Using the information provided herein, such as the
nucleotide sequences encoding at least 90-100 of the
contiguous amino acids of at least one of SEQ ID N0:3, SEQ
ID N0:4, specified fragments or variants thereof, or a
2o deposited vector comprising at least one of these sequences,
a nucleic acid molecule of the present invention encoding a
hOB-BP2h polypeptide can be obtained using well-known
methods.
Nucleic acid molecules of the present invention can be
in the form of RNA, such as mRNA, hnRNA, tRNA or any other
form, or in the form of DNA, including, but not limited to,
cDNA and genomic DNA obtained by cloning or produced
synthetically, or any combination thereof. The DNA can be
triple-stranded, double-stranded or single-stranded, or any
3o combination thereof. Any portion of at least one strand of
the DNA or RNA can be the coding strand, also known as the
sense strand, or it can be the non-coding strand, also
referred to as the anti-sense strand.
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Isolated nucleic acid molecules of the present
invention include nucleic acid molecules comprising an open
reading frame (ORF) shown in at least one of SEQ ID N0:1 and
SEQ ID N0:2; nucleic acid molecules comprising the coding
sequence for a hOB-BP2h polypeptide, and nucleic acid
molecules which comprise a nucleotide sequence substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode at least one
hOB-BP2h polypeptide as described herein. Of course, the
io genetic code is well known in the art. Thus, it would be
routine for one skilled in the art to generate such
degenerate nucleic acid variants that code for specific hOB-
BP2h polypeptides of the present invention. See, e.g.,
Ausubel, et al., supra, and such nucleic acid variants are
included in the present invention.
In another aspect, the invention provides isolated
nucleic acid molecules encoding a hOB-BP2h polypeptide
having an amino acid sequence as encoded by the cDNA clone
contained in the plasmid deposited as designated clone names
and ATCC Deposit Nos.
respectively,
deposited on
In a further embodiment, nucleic acid molecules are
provided encoding the mature hOB-BP2h polypeptide or the
full-length hOB-BP2h polypeptide lacking the N-terminal
methionine. The invention also provides an isolated nucleic
acid molecule having the nucleotide sequence shown in at
least one of SEQ ID N0:1, SEQ ID N0:2, the nucleotide
sequence of an hOB-BP2h cDNA contained in at least one of
3o the above-described deposited clones listed herein, or a
nucleic acid molecule having a sequence complementary
thereto. Such isolated molecules, particularly nucleic acid
molecules, are useful as probes for gene mapping by in situ
hybridization with chromosomes, and for detecting
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transcription, translation and expression of the hOB-BP2h
gene in human tissue, for instance, by Northern blot
analysis for mRNA detection.
Unless otherwise indicated, all nucleotide sequences
identified by sequencing a nucleic acid molecule herein can
be or were identified using an automated nucleic acid
sequences, and all amino acid sequences of polypeptides
encoded by nucleic acid molecules identified herein can be
or were identified by codon correspondence or by translation
of a nucleic acid sequence identified using method steps as
described herein or as known in the art. Therefore, as is
well known in the art that for any nucleic acid sequence
identified by this automated approach, any nucleotide
sequence identified herein may contain some errors which are
reproducibly correctable by resequencing based upon an
available or a deposited vector or host cell containing the
nucleic acid molecule using well-known methods.
Nucleotide sequences identified by automation are
typically at least about 95~ to at least about 99.999
2o identical to the actual nucleotide sequence of the sequenced
nucleic acid molecule. The actual sequence can be more
precisely identified by other approaches including manual
nucleic acid sequencing methods well known in the art. As
is also known in the art, a single insertion or deletion in
an identified nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the
nucleotide sequence such that the identified amino acid
sequence encoded by an identified nucleotide sequence will
be completely different from the amino acid sequence
actually encoded by the sequenced nucleic acid molecule,
beginning at the point of such an insertion or deletion.
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Nucleic Acid Fragments
The present invention is further directed to fragments
of the isolated nucleic acid molecules described herein. By
a fragment of an isolated nucleic acid molecule is meant a
molecule having at least 10 nucleotides of a nucleotide
sequence of a deposited cDNA or a nucleotide sequence shown
in at least one of SEQ ID N0:1 and SEQ ID N0:2, and is
intended to mean fragments at least about 10 nucleotides,
and at least about 40 nucleotides in length, which are
1o useful, inter alia as diagnostic probes and primers as
described herein. Of course, larger fragments such as at
least about 50, 100, 120, 200, 500, 1000, 1500, and 2000 in
length, are also useful according to the present invention
as are fragments corresponding to most, if not all, of the
nucleotide sequence (or the deposited cDNA) as shown in at
least one of SEQ ID N0:1 or SEQ ID N0:2. By a fragment at
least 10 nucleotides in length, for example, is intended
fragments which include 10 or more contiguous nucleotides
from the nucleotide sequence of the deposited cDNA or the
nucleotide sequence as shown in SEQ ID N0:1, SEQ ID N0:2, or
consensus sequences thereof, as determined by methods known
in the art (See e.g., Ausubel, supra, Chapter 7).
Such nucleotide fragments are useful according to the
present invention for screening DNA sequences that code for
one or more fragments of a hOB-BP2h polypeptide as described
herein. Such screening, as a non-limiting example can
include the use of so-called "DNA chips" for screening DNA
sequences of the present invention of varying lengths, as
described, e.g., in U.S. Patent Nos. 5,631,734, 5,624,711,
5,744,305, 5,770,456, 5,770,722, 5,675,443, 5,695,940,
5,710,000, 5,733,729, which are entirely incorporated herein
by reference.
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As indicated, nucleic acid molecules of the present
invention which comprise a nucleic acid encoding a hOB-BP2h
polypeptide can include, but are not limited to, those
encoding the amino acid sequence of the mature polypeptide,
by itself; the coding sequence for the mature polypeptide
and additional sequences, such as the coding sequence of at
least one signal leader or fusion peptide or. of the mature
polypeptide, with or without the aforementioned additional
coding sequences, such as at least one intron, together with
1o additional, non-coding sequences, including but not limited
to, introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and
polyadenylation signals (for example - ribosome binding and
stability of mRNA); an additional coding sequence which
codes for additional amino acids, such as those which
provide additional functionalities. Thus, the sequence
encoding a polypeptide can be fused to a marker sequence,
such as a sequence encoding a peptide that facilitates
2o purification of the fused polypeptide.
Preferred nucleic acid fragments of the present
invention also include nucleic acid molecules encoding
epitope-bearing portions of a h0B-BP2h polypeptide.
Oligoaucleotide and Polyaucleotide Probes aad Primers
In another aspect, the invention provides a
polynucleotide (either DNA or RNA) that comprises at least
about 20 nt, still more preferably at least about 30 nt, and
even more preferably at least about 30-1950 nt of a nucleic
3o acid molecule described herein. These are useful as
diagnostic probes and primers as discussed above and in more
detail below.
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By a portion of a polynucleotide of "at least 10 nt in
length," for example, is intended 10 or more contiguous
nucleotides from the nucleotide sequence of the reference
polynucleotide (e.g., at least one deposited nucleic acid or
at least one nucleotide sequence as shown in at least one of
SEQ ID N0:1 and SEQ ID N0:2).
Of course, a polynucleotide which hybridizes only to a
poly A sequence (such as the 3' terminal poly(A) of a hOB-
BP2h cDNA shown in at least one of SEQ ID N0:1, SEQ ID N0:2,
or to a complementary stretch of T (or U) resides, would not
be included in a probe of the invention, since such a
polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof
(e. g., practically any double-stranded cDNA clone).
The present invention also provides subsequences of
full-length nucleic acids. Any number of subsequences can be
obtained by reference to at least one of SEQ ID N0:1, SEQ ID
N0:2, or a complementary sequence, and using primers which
selectively amplify, under stringent conditions to: at least
2o two sites to the polynucleotides of the present invention, or
to two sites within the nucleic acid which flank and comprise
a polynucleotide of the present invention, or to a site
within a polynucleotide of the present invention and a site
within the nucleic acid which comprises it. A variety of
methods for obtaining 5' and 3' ends is well known in the
art. See, e.g., RACE (Rapid Amplification of Complementary
Ends) as described in M. A. Frohman, PCR Protocols: A Guide
to Methods and Applications, M. A. Innis, et al, Eds.,
Academic Press, Inc., San Diego, CA, pp. 28-38 (1990); see
also, U.S. Patent No. 5,470,722, and Ausubel, et al., Current
Protocols in Molecular Biology, Chapter 15, Eds., John Wiley
& Sons, N.Y. (1989-1999). Thus, the present invention
provides hOB-BP2h polynucleotides having the sequence of the
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hOB-BP2h gene, nuclear transcript, cDNA, or complementary
sequences and subsequences thereof.
Primer sequences can be obtained by reference to a
contiguous subsequence of a polynucleotide of the present
s invention. Primers are chosen to selectively hybridize,
under PCR amplification conditions, to a polynucleotide of
the present invention in an amplification mixture comprising
a genomic and cDNA library from the same species. Generally,
the primers are complementary to a subsequence of the
1o amplified nucleic acid. In some embodiments, the primers
will be constructed to anneal at their 5' terminal ends to
the codon encoding the carboxy or amino terminal amino acid
residue (or the complements thereof) of the polynucleotides
of the present invention. The primer length in nucleotides
15 is selected from the group of integers consisting of from at
least 15 to 50. Thus, the primers can be at least 15, 18,
20, 25, 30, 40, or 50 nucleotides in length or any range or
value therein. A non-annealing sequence at the 5' end of the
primer (a "tail") can be added, for example, to introduce a
2o cloning site at the terminal ends of the amplified DNA.
The amplification primers may optionally be elongated in
the 3' direction with additional contiguous or complementary
nucleotides from the polynucleotide sequences, such as at
least one of SEQ ID N0:1 and SEQ ID N0:2, from which they
25 are derived. The number of nucleotides by which the primers
can be elongated is selected from the group of integers
consisting of from at least 1 to at least 25. Thus, for
example, the primers can be elongated with an additional 1,
5, 10, or 15 nucleotides or any range or value therein.
3o Those of skill will recognize that a lengthened primer
sequence can be employed to increase specificity of binding
(i.e., annealing) to a target sequence, or to add useful
sequences, such as links or restriction sites (See e.g.,
Ausubel, supra, Chapter 15).
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The amplification products can be translated using
expression systems well known to those of skill in the art
and as discussed, infra. The resulting translation products
can be confirmed as polypeptides of the present invention by,
for example, assaying for the appropriate catalytic activity
(e.g., specific activity and substrate specificity), or
verifying the presence of one or more linear epitopes which
are specific to a polypeptide of the present invention.
Methods for protein synthesis from PCR derived templates are
1o known in the art (See e.g., Ausubel, supra, Chapters 9, 10,
15; Coligan, Current Protocols in Protein Science, supra,
Chapter 5)and available commercially. See, e.g., Amersham
Life Sciences, Inc., Catalog '97, p. 354.
Polynucleotides Hlhich Selectively Hybridize to a
Polyaucleotide as Described Herein
The present invention provides isolated nucleic acids
that hybridize under selective hybridization conditions to a
polynucleotide disclosed herein, e.g., SEQ ID N0:1 and SEQ ID
N0:2. Thus, the polynucleotides of this embodiment can be
used for isolating, detecting, and quantifying nucleic acids
comprising such polynucleotides. For example,
polynucleotides of the present invention can be used to
identify, isolate, or amplify partial or full-length clones
in a deposited library. In some embodiments, the
polynucleotides are genomic or cDNA sequences isolated, or
otherwise complementary to, a cDNA from a human or mammalian
nucleic acid library.
3o Preferably, the cDNA library comprises at least 80~
full-length sequences, preferably at least 85~ or 90~ full-
length sequences, and more preferably at least 95~ full-
length sequences. The cDNA libraries can be normalized to
increase the representation of rare sequences. Low
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stringency hybridization conditions are typically, but not
exclusively, employed with sequences having a reduced
sequence identity relative to complementary sequences.
Moderate and high stringency conditions can optionally be
employed for sequences of greater identity. Low stringency
conditions allow selective hybridization of sequences having
about 70~ sequence identity and can be employed to identify
orthologous or paralogous sequences.
Optionally, polynucleotides of this invention will
1o encode an epitope of a polypeptide encoded by the
polynucleotides described herein. The polynucleotides of this
invention embrace nucleic acid sequences that can be employed
for selective hybridization to a polynucleotide encoding a
polypeptide of the present invention.
Screening polypeptides for specific binding to
antibodies or fragments can be conveniently achieved using
peptide display libraries. This method involves the
screening of large collections of peptides for individual
members having the desired function or structure. Antibody
2o screening of peptide display libraries is well known in the
art. The displayed peptide sequences can be from 3 to 5000
or more amino acids in length, frequently from 5-100 amino
acids long, and often from about 8 to 15 amino acids long.
In addition to direct chemical synthetic methods for
generating peptide libraries, several recombinant DNA methods
have been described. One type involves the display of a
peptide sequence on the surface of a bacteriophage or cell.
Each bacteriophage or cell contains the nucleotide sequence
encoding the particular displayed peptide sequence. Such
3o methods are described in PCT Patent Publication Nos.
91/17271, 91/18980, 91/19818, and 93/08278. Other systems
for generating libraries of peptides have aspects of both in
vitro chemical synthesis and recombinant methods. See, PCT
Patent Publication Nos. 92/05258, 92/14843, and 96/19256.
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See also, U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide
display libraries, vector, and screening kits are
commercially available from such suppliers as Invitrogen
(Carlsbad, CA).
Polynucleotides Complementary to the Polynucleotides
As indicated above, the present invention provides
isolated nucleic acids comprising hOB-BP2h polynucleotides,
wherein the polynucleotides are complementary to the
polynucleotides described herein, above. As those of skill in
the art will recognize, complementary sequences base pair
throughout the entirety of their length with such
polynucleotides (i.e., have 100 sequence identity over their
entire length). Complementary bases associate through
hydrogen bonding in double-stranded nucleic acids. For
example, the following base pairs are complementary: guanine
and cytosine; adenine and thymine; and adenine and uracil.
(See, e.g., Ausubel, supra, Chapter 67; or Sambrook, supra)
2o Construction of Nucleic Acids
The isolated nucleic acids of the present invention can
be made using (a) standard recombinant methods, (b) synthetic
techniques, (c) purification techniques, or combinations
thereof, as well known in the art.
The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For
example, a multi-cloning site comprising one or more
endonuclease restriction sites may be inserted into the
nucleic acid to aid in isolation of the polynucleotide.
3o Also, translatable sequences may be inserted to aid in the
isolation of the translated polynucleotide of the present
invention. For example, a hexa-histidine marker sequence
provides a convenient means to purify the proteins of the
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present invention. The nucleic acid of the present invention
- excluding the polynucleotide sequence - is optionally a
vector, adapter, or linker for cloning and expression of a
polynucleotide of the present invention.
Additional sequences may be added to such cloning and
expression sequences to optimize their function in cloning
and expression, to aid in isolation of the polynucleotide, or
to improve the introduction of the polynucleotide into a
cell. Typically, the length of a nucleic acid of the present
1o invention less the length of its polynucleotide of the
present invention is less than 20 kilobase pairs, often less
than 15 kb, and frequently less than 10 kb. Use of cloning
vectors, expression vectors, adapters, and linkers is well
known in the art. (See, e.g., Ausubel, supra, Chapters 1-5;
or Sambrook, supra).
Recombinant Methods for Constructing Nhcleic Acids
The isolated nucleic acid compositions of this
invention, such as RNA, cDNA, genomic DNA, or a hybrid
2o thereof, can be obtained from biological sources using any
number of cloning methodologies known to those of skill in
the art. In some embodiments, oligonucleotide probes that
selectively hybridize, under stringent conditions, to the
polynucleotides of the present invention are used to identify
the desired sequence in a cDNA or genomic DNA library. lnlhile
isolation of RNA, and construction of cDNA and genomic
libraries is well known to those of ordinary skill in the
art. (See, e.g., Ausubel, supra, Chapters 1-7; or Sambrook,
supra)
Nucleic Acid Screening and Isolation Methods
A cDNA or genomic library can be screened using a probe
based upon the sequence of a polynucleotide of the present
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invention, such as those disclosed herein. Probes may be
used to hybridize with genomic DNA or cDNA sequences to
isolate homologous genes in the same or different organisms.
Those of skill in the art will appreciate that various
degrees of stringency of hybridization can be employed in the
assay; and either the hybridization or the wash medium can be
stringent. As the conditions for hybridization become more
stringent, there must be a greater degree of complementarity
between the probe and the target for duplex formation to
occur. Temperature, ionic strength, pH and the presence of a
partially denaturing solvent such as formamide can control
the degree of stringency. Changing the polarity of the
reactant solution through, for example, manipulation of the
concentration of formamide within the range of 0~ to 50~
conveniently varies the stringency of hybridization. The
degree of complementarity (sequence identity) required for
detectable binding will vary in accordance with the
stringency of the hybridization medium and wash medium. The
degree of complementarity will optimally be 100; however, it
2o should be understood that minor sequence variations in the
probes and primers may be compensated for by reducing the
stringency of the hybridization and wash medium.
Methods of amplification of RNA or DNA are well known
in the art and can be used according to the present
invention without undue experimentation, based on the
teaching and guidance presented herein.
Known methods of DNA or RNA amplification include, but
are not limited to, polymerase chain reaction (PCR) and
related amplification processes (see, e.g., U.S. Patent Nos.
4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et
al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 to
Innis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis;
5,066,584 to Gyllensten, et al; 4,889,818 to Gelfand, et al;
4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134
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to Ringold) and RNA mediated amplification which uses anti-
sense RNA to the target sequence as a template for double-
stranded DNA synthesis (U. S. Patent No. 5,130,238 to Malek,
et al, with the tradename NASBA), the entire contents of
which are herein incorporated by reference. (See, e.g.,
Ausubel, supra, Chapter 15; or Sambrook, supra)
For instance, polymerase chain reaction (PCR) technology
can be used to amplify the sequences of polynucleotides of
the present invention and related genes directly from genomic
DNA or cDNA libraries. PCR and other in vitro amplification
methods may also be useful, for example, to clone nucleic
acid sequences that code for proteins to be expressed, to
make nucleic acids to use as probes for detecting the
presence of the desired mRNA in samples, for nucleic acid
sequencing, or for other purposes. Examples of techniques
sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, Sambrook, and
Ausubel (e. g., Chapter 15) supra, as well as Mullis, et al.,
U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR
Protocols A Guide to Methods and Applications, Eds., Academic
Press Inc., San Diego, CA (1990). Commercially available
kits for genomic PCR amplification are known in the art. See,
e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene
32 protein (Boehringer Mannheim) can be used to improve yield
of long PCR products.
Synthetic Methods for Constructing Nucleic Acids
The isolated nucleic acids of the present invention can
also be prepared by direct chemical synthesis by methods such
3o as the phosphotriester method of Narang, et al., Meth.
Enzymol. 68:90-99 (1979); the phosphodiester method of Brown,
et al., Meth. Enzymol. 68:109-151 (1979); the
diethylphosphoramidite method of Beaucage, et al., Tetra.
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Letts. 22:1859-1862 (1981); the solid phase phosphoramidite
triester method described by Beaucage and Caruthers, Tetra.
Letts. 22(20):1859-1862 (1981), e.g., using an automated
synthesizer; e.g., as described in Needham-VanDevanter, et
al., Nucleic Acids Res. 12:6159-6168 (1984); and the solid
support method of U.S. Patent No. 4,458,066. Chemical
synthesis generally produces a single-stranded
oligonucleotide, which may be converted into double-stranded
DNA by hybridization with a complementary sequence, or by
1o polymerization with a DNA polymerase using the single strand
as a template. One of skill in the art will recognize that
while chemical synthesis of DNA can be limited to sequences
of about 100 or more bases, longer sequences may be obtained
by the ligation of shorter sequences.
Recombinant Expression Cassettes
The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence of the present invention,
2o for example a cDNA or a genomic sequence encoding a full-
length polypeptide of the present invention, can be used to
construct a recombinant expression cassette which can be
introduced into at least one desired host cell. A
recombinant expression cassette will typically comprise a
polynucleotide of the present invention operably linked to
transcriptional initiation regulatory sequences that will
direct the transcription of the polynucleotide in the
intended host cell.
Both heterologous and non-heterologous (i.e.,
3o endogenous) promoters can be employed to direct expression of
the nucleic acids of the present invention. These promoters
can also be used, for example, in recombinant expression
cassettes to drive expression of antisense nucleic acids to
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reduce, increase, or alter hOB-BP2h content and composition
in a desired tissue.
In some embodiments, isolated nucleic acids which serve
as promoter or enhancer elements can be introduced in the
appropriate position (generally upstream) of a non-
heterologous form of a polynucleotide of the present
invention so as to up or down regulate expression of a
polynucleotide of the present invention. For example,
endogenous promoters can be altered in vivo or in vitro by
1o mutation, deletion and substitution.
A polynucleotide of the present invention can be
expressed in either sense or anti-sense orientation as
desired. It will be appreciated that control of gene
expression in either sense or anti-sense orientation can have
a direct impact on the observable characteristics.
Another method of suppression is sense suppression.
Introduction of nucleic acid configured in the sense
orientation has been shown to be an effective means by which
to block the transcription of target genes.
2o A variety of cross-linking agents, alkylating agents and
radical generating species as pendant groups on
polynucleotides of the present invention can be used to bind,
label, detect and cleave nucleic acids. Knorre, et al.,
Biochimie 67:785-789 (1985); Vlassov, et al., Nucleic Acids
Res. 14:4065-4076 (1986); Iverson and Dervan, J. Am. Chem.
Soc. 109:1241-1243 (1987); Meyer, et al., J. Am. Chem. Soc.
111:8517-8519 (1989); Lee, et al., Biochemistry 27:3197-3203
(1988); Home, et al., J. Am. Chem. Soc. 112:2435-2437 (1990);
Webb and Matteucci, J. Am. Chem. Soc. 108:2764-2765 (1986);
3o Nucleic Acids Res. 14:7661-7674 (1986); Feteritz, et al., J.
Am. Chem. Soc. 113:4000 (1991). Various compounds to bind,
detect, label, and cleave nucleic acids are known in the art.
See, for example, U.S. Patent Nos. 5,543,507; 5,672,593;
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5,484,908; 5,256,648; and 5,681941, each entirely
incorporated herein by reference.
VECTORS AND HOST CELLS
The present invention also relates to vectors that
include isolated nucleic acid molecules of the present
invention, host cells that are genetically engineered with
the recombinant vectors, and the production of hOB-BP2h
polypeptides or fragments thereof by recombinant techniques,
1o as is well known in the art. See, e.g., Sambrook, et al.,
supra; Ausubel, supra, Chapters 1-9, each entirely
incorporated herein by reference.
The polynucleotides can optionally be joined to a
vector containing a selectable marker for propagation in a
host. Generally, a plasmid vector is introduced in a
precipitate, such as a calcium phosphate precipitate, or in
a complex with a charged lipid. If the vector is a virus,
it can be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter,
the E. coli lac, trp and tac promoters, the SV40 early and
late promoters and promoters of retroviral LTRs, or any
other suitable promoter. The skilled artisan will know
other suitable promoters. The expression constructs will
further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome-
binding site for translation. The coding portion of the
mature transcripts expressed by the constructs will
preferably include a translation initiating at the beginning
and a termination codon (e. g., UAA, UGA or UAG)
appropriately positioned at the end of the mRNA to be
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translated, with VAA and VAG preferred for mammalian or
eukaryotic cell expression.
Expression vectors will preferably include at least one
selectable marker. Such markers include, e.g.,
dihydrofolate reductase, ampicillin (G418), hygromycin or
neomycin resistance for eukaryotic cell culture, and
tetracycline or ampicillin resistance genes for culturing in
E. coli and other bacteria or prokaryotics. Representative
examples of appropriate hosts include, but are not limited
1o to, bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast
cells; insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in
the art. Vectors preferred for use in bacteria include
pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNHBA, pNHl6a,
pNHl8A, pNH46A, available from Stratagene; and ptrc99a,
2o pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
Preferred eucaryotic vectors include pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan. See, e.g., Ausubel, supra, Chapter 1; Coligan,
Current Protocols in Protein Science, supra, Chapter 5.
Introduction of a vector construct into a host cell can
be effected by calcium phosphate transfection, DEAF-dextran
mediated transfection, cationic lipid-mediated transfection,
3o electroporation, transduction, infection or other methods.
Such methods are described in many standard laboratory
manuals, such as Sambrook, supra, Chapters 1-4 and 16-18;
Ausubel, supra, Chapters 1, 9, 13, 15, 16.
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Polypeptide(s) of the present invention can be
expressed in a modified form, such as a fusion protein, and
can include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of
additional amino acids, particularly charged amino acids,
can be added to the N-terminus of a polypeptide to improve
stability and persistence in the host cell, during
purification, or during subsequent handling and storage.
Also, peptide moieties can be added to a polypeptide to
1o facilitate purification. Such regions can be removed prior
to final preparation of a polypeptide. Such methods are
described in many standard laboratory manuals, such as
Sambrook, supra, Chapters 17 and 18; Ausubel, supra,
Chapters 16, 17 and 18.
Expression of Proteins in Host Cells
Using nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell, such as bacteria, yeast, insect, or
2o mammalian cells. The cells produce the protein in a non-
natural condition (e. g., in quantity, composition, location,
and time), because they have been genetically altered through
human intervention to do so.
It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available
for expression of a nucleic acid encoding a protein of the
present invention. No attempt to describe in detail the
various methods known for the expression of proteins in
prokaryotes or eukaryotes will be made.
3o In brief summary, the expression of isolated nucleic
acids encoding a protein of the present invention will
typically be achieved by operably linking, for example, the
DNA or cDNA to a promoter (which is either constitutive or
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inducible) followed by incorporation into an expression
vector. The vectors can be suitable for replication and
integration in either prokaryotes or eukaryotes. Typical
expression vectors contain transcription and translation
terminators, initiation sequences and promoters useful for
regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a
cloned gene, it is desirable to construct expression vectors
which contain, at the minimum, a strong promoter to direct
1o transcription, a ribosome binding site for translational
initiation, and a transcription/translation terminator. One
of skill would recognize that modifications can be made to a
protein of the present invention without diminishing its
biological activity. Some modifications may be made to
facilitate the cloning, expression, or incorporation of the
targeting molecule into a fusion protein. Such modifications
are well known to those of skill in the art and include, for
example, a methionine added at the amino terminus to provide
an initiation site, or additional amino acids (e. g., poly
2o His) placed on either terminus to create conveniently located
restriction sites or termination codons or purification
sequences.
Alternatively, nucleic acids of the present invention
can be expressed in a host cell by turning on (by
manipulation) in a host cell that contains endogenous DNA
encoding a polypeptide of the present invention. Such
methods are well known in the art, e.g., as described in US
patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761,
entirely incorporated herein by reference.
Expression in Prokaryotes
Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various
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strains of E. coli; however, other microbial strains may also
be used. Commonly used prokaryotic control sequences which
are defined herein to include promoters for transcription
initiation, optionally with an operator, along with ribosome
binding site sequences, include such commonly used promoters
as the beta lactamase (penicillinase) and lactose (lac)
promoter systems (Chang, et al., Nature 198:1056 (1977)), the
tryptophan (trp) promoter system (Goeddel, et al., Nucleic
Acids Res. 8:4057 (1980)) and the lambda derived P L promoter
1o and N-gene ribosome binding site (Shimatake, et al., Nature
292:128 (1981)). The inclusion of selection markers in DNA
vectors transfected in E. coli is also useful. Examples of
such markers include genes specifying resistance to
ampicillin, tetracycline, or chloramphenicol.
The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of
plasmid or phage origin. Appropriate bacterial cells are
infected with phage vector particles or transfected with
naked phage vector DNA. If a plasmid vector is used, the
2o bacterial cells are transformed with the plasmid vector DNA.
Expression systems for expressing a protein of the present
invention are available using Bacillus sp. and Salmonella
(Palva, et al., Gene 22:229-235 (1983); Mosbach, et al.,
Nature 302:543-545 (1983)). See, e.g., Ausubel, supra,
Chapters 1-3, 16(Sec.1); and Coligan, supra, Current
Protocols in Protein Science, Units 5.1, 6.1-6.7.
Expression in Eukaryotes
A variety of eukaryotic expression systems such as
3o yeast, insect cell lines, plant and mammalian cells, are
known to those of skill in the art. As explained briefly
below, a nucleic acid of the present invention can be
expressed in these eukaryotic systems.
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Synthesis of heterologous proteins in yeast is well
known. F. Sherman, et al., Methods in Yeast Genetics, Cold
Spring Harbor Laboratory (1982) is a well-recognized work
describing the various methods available to produce the
protein in yeast. Two widely utilized yeast for production
of eukaryotic proteins are Saccharomyces cerevisiae and
Pichia pastoris. Vectors, strains, and protocols for
expression in Saccharomyces and Pichia are known in the art
and available from commercial suppliers (e. g., Invitrogen).
to Suitable vectors usually have expression control sequences,
such as promoters, including 3-phosphoglycerate kinase or
alcohol oxidase, and an origin of replication, termination
sequences and the like as desired.
A protein of the present invention, once expressed, can
be isolated from yeast by lysing the cells and applying
standard protein isolation techniques to the lysates. The
monitoring of the purification process can be accomplished by
using Western blot techniques or radioimmunoassay of other
standard immunoassay techniques.
2o The sequences encoding proteins of the present invention
can also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian,
insect, or plant origin. Illustrative of cell cultures
useful for the production of the peptides are mammalian
cells. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions may
also be used. A number of suitable host cell lines capable
of expressing intact proteins have been developed in the art,
and include the HEK293, BHK21, and CHO cell lines.
3o Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a
promoter (e.g., the CMV promoter, a HSV tk promoter or pgk
(phosphoglycerate kinase) promoter), an enhancer (Queen, et
al., Immunol. Rev. 89:49 (1986)), and processing information
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sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites (e.g., an SV40 large T Ag poly A
addition site), and transcriptional terminator sequences.
Other animal cells useful for production of proteins of the
present invention are available, for instance, from the
American Type Culture Collection Catalogue of Cell Lines and
Hybridomas (7th edition, 1992).
Appropriate vectors for expressing proteins of the
present invention in insect cells are usually derived from
1o the SF9 baculovirus. Suitable insect cell lines include
mosquito larvae, silkworm, armyworm, moth and Drosophila cell
lines such as a Schneider cell line (See Schneider, J.
Embryol. Exp. Morphol. 27:353-365 (1987).
As with yeast, when higher animal or plant host cells
are employed, polyadenlyation or transcription terminator
sequences are typically incorporated into the vector. An
example of a terminator sequence is the polyadenlyation
sequence from the bovine growth hormone gene. Sequences for
accurate splicing of the transcript may also be included. An
2o example of a splicing sequence is the VP1 intron from SV40
(Sprague, et al., J. Virol. 45:773-781 (1983)).
Additionally, gene sequences to control replication in the
host cell may be incorporated into the vector such as those
found in bovine papilloma virus type-vectors. M. Saveria-
Campo, Bovine Papilloma Virus DNA, a Eukaryotic Cloning
Vector in DNA Cloning Vol. II, a Practical Approach, D. M.
Glover, Ed., IRL Press, Arlington, VA, pp. 213-238 (1985).
Protein Purification
3o A h0B-BP2h polypeptide can be recovered and purified
from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
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phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably,
high performance liquid chromatography ("HPLC") is employed
for purification. Polypeptides of the present invention
include naturally purified products, products of chemical
synthetic procedures, and products produced by recombinant
techniques from a prokaryotic or eucaryotic host, including,
for example, bacterial, yeast, higher plant, insect and
1o mammalian cells. Depending upon the host employed in a
recombinant production procedure, the polypeptides of the
present invention can be glycosylated or can be non-
glycosylated. In addition, polypeptides of the invention
can also include an initial modified methionine residue, in
some cases as a result of host-mediated processes. Such
methods are described in many standard laboratory manuals,
such as Sambrook, supra, Chapters 17.37-17.42; Ausubel,
supra, Chapters 10, 12, 13, 16, 18 and 20.
hOB-BP2h POLYPEPTIDES AND FRAGMENTS AND VARIANTS
2o The invention further provides isolated hOB-BP2h
polypeptides having fragments or specified variants of the
amino acid sequence encoded by the deposited cDNAs, or the
amino acid sequences in SEQ ID N0:3, 4, or 5.
The isolated proteins of the present invention comprise
a polypeptide encoded by any one of the polynucleotides of
the present invention as discussed more fully, supra, or
polypeptides which are specified fragments or variants
thereof .
Exemplary polypeptide sequences are provided in SEQ ID
3o N0:3, 4, or 5. The proteins of the present invention, or
variants thereof, can comprise any number of contiguous amino
acid residues from a polypeptide of the present invention,
wherein that number is selected from the group of integers
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consisting of from 90-100 of the number of contiguous
residues in a full-length hOB-BP2h polypeptide. Optionally,
this subsequence of contiguous amino acids is at least 50,
60, 70, 80, or 90 amino acids in length. Further, the number
of such subsequences can be any integer selected from the
group consisting of from 1 to 20, such as 2, 3, 4, or 5.
As those of skill will appreciate, the present invention
includes biologically active polypeptides of the present
invention (i.e., enzymes). Biologically active polypeptides
have a specific activity at least 20~, 30~, or 40~, and
preferably at least 50~, 60~, or 70~, and most preferably at
least 80~, 90~, or 95~-1000 of that of the native (non-
synthetic), endogenous polypeptide. Further, the substrate
specificity (e. g., k~at/Rm) is optionally substantially
similar to the native (non-synthetic), endogenous
polypeptide. Typically, the K~, will be at least 30~, 40~, or
50~, that of the native (non-synthetic), endogenous
polypeptide; and more preferably at least 60~, 70~, 80~, or
90~-1000. Methods of assaying and quantifying measures of
enzymatic activity and substrate specificity, are well known
to those of skill in the art.
Generally, the polypeptides of the present invention
will, when presented as an immunogen, elicit production of an
antibody specifically reactive to a polypeptide of the
present invention encoded by a polynucleotide of the present
invention as described, supra. Exemplary polypeptides
include those which are full-length, such as those disclosed
herein. Further, the proteins of the present invention will
not bind to antisera raised against a polypeptide of the
3o present invention which has been fully immunosorbed with the
same polypeptide. Immunoassays for determining binding are
well known to those of skill in the art. A preferred
immunoassay is a competitive immunoassay as discussed, infra.
Thus, the proteins of the present invention can be employed
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as immunogens for constructing antibodies immunoreactive to a
protein of the present invention for such exemplary utilities
as immunoassays or protein purification techniques.
A hOB-BP2h polypeptide of the present invention can
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human
manipulation, as specified herein.
Of course, the number of amino acid substitutions a
skilled artisan would make depends on many factors,
1o including those described above. Generally speaking, the
number of amino acid substitutions, insertions or deletions
for any given hOB-BP2h polypeptide will not be more than 40,
30, 20, 10, 5, or 3, such as 1-30 or any range or value
therein, as specified herein.
Amino acids in a hOB-BP2h polypeptide of the present
invention that are essential for function can be identified
by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and
Wells, Science 244:1081-1085 (1989)). The latter procedure
2o introduces single alanine mutations at every residue in the
molecule. The resulting mutant molecules are then tested
for biological activity. Sites that are critical for
ligand-protein binding can also be identified by structural
analysis such as crystallization, nuclear magnetic resonance
or photoaffinity labeling (Smith, et al., J. Mol. Biol.
224:899-904 (1992) and de Vos, et al., Science 255:306-312
(1992)).
hOB-BP2h polypeptides of the present invention can
include but are not limited to, at least one selected from
the extracellular domain, intracellular domain,
transmembrane domain, and active domain of SEQ ID N0:3, 4,
or 5.
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A hOB-BP2h polypeptide
can further comprise a
polypeptide of at least one 512 or 639 contiguous amino
of
acids of SEQ ID N0:3 or SEQ N0:4 , respectively.
ID
A hOB-BP2h polypeptide rtherincludes an amino acid
fu
sequence selected from more of SEQ ID N0:3, 4, or
one or 5.
Non-limiting mutants that can enhance or maintain at
least one of the listed activities include, but are not
limited to, any of the olypeptides,
above p further
comprising at least one mutation
corresponding
to
at
least
10one substitution, insertion deletion
or selected
from
the
group consisting of 3P, 4L, , 11W, 15L, 16Q, 17E, 18K,
8P 9L,
19P, 20V, 21Y, 22E, 23L, 24Q, 27K, 30T, 32Q, 37V, 38L, 47W,
48R, 495, 51Y, 525, 54P, 56L, 58V, 70A, 71E, 72V, 77N, 78P,
79D, 81R, 83K, 84P, 85E, 87Q, 91R, 93L, 96V, 97Q, 99K, 104S,
151066, 1098, 111E, 113T, 1146, 1155,1248, 125D, 127K, 129S,
130Y, 131Q, 132Q, 133N, 134K, 135L,136N, 138E, 141V, 143S,
143I, 144F, 144E, 145T, 210N, and 52A of SEQ ID N0:3, or
2
3P, 4L, 8P, 9L, 11W, 15L, 17E, 18K, 19P, 20V, 21Y, 22E,
16Q,
23L, 24Q, 27K, 30T, 32Q, 37V, 38L, 47W, 48R, 49S, 51Y, 525,
2054P, 56L, 58V, 70A, 71E, 72V, 77N, 78P, 79D, 81R, 83K, 84P,
85E, 87Q, 91R, 93L, 96V, 97Q, 99K, 1045, 1066, 1098, 111E,
113T, 1146, 1155, 1248, 125D, 127K,1295, 130Y, 131Q, 132Q,
133N, 134K, 135L, 136N, 138E, 141A,143T, 143I, 144Q, 144E,
145K, 152D, 194D, 3865, 387L, 389Q,4058, 408F, 4098, 4118,
25417C, 4198, 421E, 423K, 424P, 4326,435K, 437N, 4386, 450I,
458D, 460K, 461V, 4625, 464K, 468I,469Y, 471S, and 476V
of SEQ ID N0:4.
Antigenic/Epitope Comprising hOB-BP2h Peptide and
30 Polypeptides
In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a
polypeptide of the invention according to methods well known
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in the art. See, e.g., Colligan, et al,. ed., Current
Protocols in Immunology, Greene Publishing, NY (1993-1998),
Ausubel, supra, each entirely incorporated herein by
reference.
The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide described
herein. An "immunogenic epitope" can be defined as a part
of a polypeptide that elicits an antibody response when the
whole polypeptide is the immunogen. On the other hand, a
1o region of a polypeptide molecule to which an antibody can
bind is defined as an "antigenic epitope." The number of
immunogenic epitopes of a polypeptide generally is less than
the number of antigenic epitopes. See, for instance,
Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983) .
As to the selection of peptides or polypeptides bearing
an antigenic epitope (i.e., that contain at least a portion
of a region of a polypeptide molecule to which an antibody
can bind), it is well known in the art that relatively short
2o synthetic peptides that mimic part of a polypeptide sequence
are routinely capable of eliciting an antiserum that reacts
with the partially mimicked polypeptide. See, for instance,
J. G. Sutcliffe, et al., "Antibodies that react with
preidentified sites on polypeptides," Science 219:660-666
( 1983 ) .
Antigenic epitope-bearing peptides and polypeptides of
the invention are useful to raise antibodies, including
monoclonal antibodies, or screen antibodies, including
fragments or single chain antibodies, that bind specifically
3o to a polypeptide of the invention. See, for instance,
Wilson, et al., Cell 37:767-778 (1984) at 777. Antigenic
epitope-bearing peptides and polypeptides of the invention
preferably contain a sequence of at least five, more
preferably at least nine, and most preferably between at
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least about 15 to about 30 amino acids contained within the
amino acid sequence of a polypeptide of the invention.
The epitope-bearing peptides and polypeptides of the
invention can be produced by any conventional means. R. A.
Houghten, "General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual
amino acids," Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No.
4,631,211 to Houghten, et al. (1986).
As one of skill in the art will appreciate, hOB-BP2h
polypeptides of the present invention and the epitope-
bearing fragments thereof described above can be combined
with parts of the constant domain of immunoglobulins (Fc),
resulting in chimeric polypeptides. These fusion proteins
facilitate purification and show an increased half-life in
vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-
2o polypeptide and various domains of the constant regions of
the heavy or light chains of mammalian immunoglobulins (EPA
394,827; Traunecker, et al., Nature 331:84-86 (1988)).
Fusion proteins that have a disulfide-linked dimeric
structure due to the IgG part can also be more efficient in
binding and neutralizing other molecules than the monomeric
hOB-BP2h polypeptide or polypeptide fragment alone
(Fountoulakis, et al., J. Biochem. 270:3958-3964 (1995)).
Production of Antibodies
The polypeptides of this invention and fragments
thereof may be used in the production of antibodies. The
term "antibody" as used herein describes antibodies,
fragments of antibodies (such as, but not limited, to Fab,
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Fab', Fab2', and Fv fragments), and modified versions
thereof, as well known in the art (e. g., chimeric,
humanized, recombinant, veneered, resurfaced or CDR-grafted)
such antibodies are capable of binding antigens of a similar
nature as the parent antibody molecule from which they are
derived. The instant invention also encompasses single
chain polypeptide binding molecules.
The production of antibodies, both monoclonal and
polyclonal, in animals is well known in the art. See, e.g.,
1o Colligan, supra, entirely incorporated herein by reference.
Single chain antibodies and libraries thereof are yet
another variety of genetically engineered antibody
technology that is well known in the art. (See, e.g., R. E.
Bird, et al., Science 242:423-426 (1988); PCT Publication
Nos. WO 88/01649, WO 90/14430, and WO 91/10737. Single
chain antibody technology involves covalently joining the
binding regions of heavy and light chains to generate a
single polypeptide chain. The binding specificity of the
intact antibody molecule is thereby reproduced on a single
polypeptide chain.
Antibodies included in this invention are useful in
diagnostics, therapeutics or in diagnostic/therapeutic
combinations.
The polypeptides of this invention or suitable
fragments thereof can be used to generate polyclonal or
monoclonal antibodies, and various inter-species hybrids, or
humanized antibodies, or antibody fragments, or single-chain
antibodies. The techniques for producing antibodies are well
known to skilled artisans. (See, e.g., Colligan supra;
3o Monoclonal Antibodies: Principles & Applications, Ed. J. R.
Birch & E. S. Lennox, Wiley-Liss (1995).
A polypeptide used as an immunogen may be modified or
administered in an adjuvant, by subcutaneous or
intraperitoneal injection into, for example, a mouse or a
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rabbit. For the production of monoclonal antibodies, spleen
cells from immunized animals are removed, fused with myeloma
or other suitable known cells, and allowed to become
monoclonal antibody producing hybridoma cells in the manner
known to the skilled artisan. Hybridomas that secrete a
desired antibody molecule can be screened by a variety of
well known methods, for example ELISA assay, Western blot
analysis, or radioimmunoassay (Lutz, et al. Exp. Cell Res.
175:109-124 (1988); Monoclonal Antibodies: Principles &
l0 Applications, Ed. J. R. Birch & E. S. Lennox, Wiley-Liss
(1995); Colligan, supra).
For some applications labeled antibodies are desirable.
Procedures for labeling antibody molecules are widely known,
including for example, the use of radioisotopes, affinity
labels, such as biotin or avidin, enzymatic labels, for
example horseradish peroxidase, and fluorescent labels, such
as FITC or rhodamine (See, e.g., Colligan, supra).
Labeled antibodies are useful for a variety of
diagnostic applications. In one embodiment the present
2o invention relates to the use of labeled antibodies to detect
the presence of a hOB-BP2h polypeptide. Alternatively, the
antibodies could be used in a screen to identify potential
modulators of a hOB-BP2h polypeptide. For example, in a
competitive displacement assay, the antibody or compound to
be tested is labeled by any suitable method. Competitive
displacement of an antibody from an antibody-antigen complex
by a test compound such that a test compound-antigen complex
is formed provides a method for identifying compounds that
bind HPLFP:
Transgenics and Chimeric Non-Human Mammals
The present invention is also directed to a transgenic
non-human eukaryotic animal (preferably a rodent, such as a
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mouse) the germ cells and somatic cells of which contain
nucleic acid genomic DNA according to the present invention
which codes for at least one hOB-BP2h polypeptide. At least
one hOB-BP2h nucleic acid can be introduced into the animal
to be made transgenic, or an ancestor of the animal, at an
embryonic stage, preferably the 1-1000 cell or oocyte, stage,
and preferably not later than about the 64-cell stage. The
term "transgene," as used herein, means a gene which is
incorporated into the genome of the animal and is expressed
1o in the animal, resulting in the presence of at least one hOB-
BP2h polypeptide in the transgenic animal.
There are several means by which such a hOB-BP2h nucleic
acid can be introduced into a cell or genome of the animal
embryo so as to be chromosomally incorporated and expressed
according to known methods.
Chimeric non-human mammals in which fewer than all of
the somatic and germ cells contain the a hOB-BP2h polypeptide
nucleic acid of the present invention, such as animals
produced when fewer than all of the cells of the morula are
2o transfected in the process of producing the transgenic
animal, are also intended to be within the scope of the
present invention.
Chimeric non-human mammals having human cells or tissue
engrafted therein are also encompassed by the present
invention, which may be used for testing expression of at
least one hOB-BP2h polypeptide in human tissue and for
testing the effectiveness of therapeutic and diagnostic
agents associated with delivery vectors which preferentially
bind to a hOB-BP2h polypeptide of the present invention.
3o Methods for providing chimeric non-human mammals are
provided, e.g., in U.S. Serial Nos. 07/508,225, 07/518,748,
07/529,217, 07/562,746, 07/596,518, 07/574,748, 07/575,962,
07/207,273, 07/241,590 and 07/137,173, which are entirely
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incorporated herein by reference, for their description of
how to engraft human cells or tissue into non-human mammals.
The techniques described in Leder, U.S. Patent No.
4,736,866 (hereby entirely incorporated by reference) for
producing transgenic non-human mammals may be used for the
production of a transgenic non-human mammal of the present
invention. The various techniques described in U.S. patent
Nos. 5,454,807, 5,073,490, 5,347,075 and 4,736,866, the
entire contents of which are hereby incorporated by
1o reference, may also be used.
Animals carrying at least one hOB-BP2h polypeptide and
nucleic acid can be used to test compounds or other treatment
modalities which may prevent, suppress or cure a pathology
relating to at least one hOB-BP2h polypeptide or hOB-BP2h
nucleic acid. Such transgenic animals will also serve as a
model for testing of diagnostic methods for the same
diseases. Transgenic animals according to the present
invention can also be used as a source of cells for cell
culture.
Having generally described the invention, the same will
be more readily understood by reference to the following
examples, which are provided by way of illustration and are
not intended as limiting.
Example 1: Expression and Purification of an hOB-BP2h
Polypeptide in E. coli
The bacterial expression vector pQE60 is used for
bacterial expression in this example. (QIAGEN, Inc.,
Chatsworth, CA). pQE60 encodes ampicillin antibiotic
3o resistance ("Ampr") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, a ribosome
binding site ("RBS"), six codons encoding histidine residues
that allow affinity purification using nickel-nitrilo-tri-
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acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc.,
and suitable single restriction enzyme cleavage sites.
These elements are arranged such that a DNA fragment
encoding a polypeptide can be inserted in such a way as to
produce that polypeptide with the six His residues (i.e., a
"6 X His tag") covalently linked to the carboxyl terminus of
that polypeptide. However, a polypeptide coding sequence
can optionally be inserted such that translation of the six
His codons is prevented and, therefore, a polypeptide is
1o produced with no 6 X His tag.
The nucleic acid sequence encoding the desired portion
of a hOB-BP2h polypeptide lacking the hydrophobic leader
sequence is amplified from the deposited cDNA clone using
PCR oligonucleotide primers (based on the sequences
presented, e.g., in at least one of SEQ ID N0:1 and SEQ ID
N0:2), which anneal to the amino terminal encoding DNA
sequences of the desired portion of a hOB-BP2h polypeptide
and to sequences in the deposited construct 3' to the cDNA
coding sequence. Additional nucleotides containing
2o restriction sites to facilitate cloning in the pQE60 vector
are added to the 5' and 3' sequences, respectively.
For cloning a hOB-BP2h polypeptide, the 5' and 3'
primers have nucleotides corresponding or complementary to a
portion of the coding sequence of a h0B-BP2h, e.g., as
presented in at least one of SEQ ID N0:1 and SEQ ID N0:2,
according to known method steps. One of ordinary skill in
the art would appreciate, of course, that the point in a
polypeptide coding sequence where the 5' primer begins can
be varied to amplify a desired portion of the complete
3o polypeptide shorter or longer than the mature form.
The amplified hOB-BP2h nucleic acid fragments and the
vector pQE60 are digested with appropriate restriction
enzymes and the digested DNAs are then ligated together.
Insertion of the hOB-BP2h DNA into the restricted pQE60
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vector places a hOB-BP2h polypeptide coding region including
its associated stop codon downstream from the 2PTG-inducible
promoter and in-frame with an initiating AUG codon. The
associated stop codon prevents translation of the six
histidine codons downstream of the insertion point.
The ligation mixture is transformed into competent E.
coli cells using standard procedures such as those described
in Sambrook, et al., 1989; Ausubel, 1987-1998. E. coli
strain M15/rep4, containing multiple copies of the plasmid
1o pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which
is only one of many that are suitable for expressing hOB-
BP2h polypeptide, is available commercially from QIAGEN,
Inc. Transformants are identified by their ability to grow
on LB plates in the presence of ampicillin and kanamycin.
Plasmid DNA is isolated from resistant colonies and the
identity of the cloned DNA confirmed by restriction
analysis, PCR and DNA sequencing.
2o Clones containing the desired constructs are grown
overnight ("0/N") in liquid culture in LB media supplemented
with both ampicillin (100 ~g/ml) and kanamycin (25 ~g/ml).
The O/N culture is used to inoculate a large culture, at a
dilution of approximately 1:25 to 1:250. The cells are
grown to an optical density at 600 nm ("OD600") of between
0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG") is
then added to a final concentration of 1 mM to induce
transcription from the lac repressor sensitive promoter, by
inactivating the lacI repressor. Cells subsequently are
3o incubated further for 3 to 4 hours. Cells then are
harvested by centrifugation.
The cells are then stirred for 3-4 hours at 4°C in 6M
guanidine-HC1, pH8. The cell debris is removed by
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centrifugation, and the supernatant containing the hOB-BP2h
is dialyzed against 50 mM Na-acetate buffer pH6,
supplemented with 200 mM NaCl. Alternatively, a polypeptide
can be successfully refolded by dialyzing it against 500 mM
NaCl, 20~ glycerol, 25 mM Tris/HC1 pH7.4, containing
protease inhibitors.
If insoluble protein is generated, the protein is made
soluble according to known method steps. After renaturation
the polypeptide is purified by ion exchange, hydrophobic
l0 interaction and size exclusion chromatography.
Alternatively, an affinity chromatography step such as an
antibody column is used to obtain pure hOB-BP2h polypeptide.
The purified polypeptide is stored at 4°C or frozen at -
40°C
to -120°C.
Example 2: Cloning and Expression of an hOB-BP2h
Polypeptide in a Baculovirus Expression System
In this illustrative example, the plasmid shuttle
vector pA2 GP is used to insert the cloned DNA encoding the
2o mature polypeptide into a baculovirus to express a hOB-BP2h
polypeptide, using a baculovirus leader and standard methods
as described in Summers, et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures,
Texas Agricultural Experimental Station Bulletin No. 1555
(1987). This expression vector contains the strong
polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the secretory signal
peptide (leader) of the baculovirus gp67 polypeptide and
convenient restriction sites such as BamHI, Xba I and
3o Asp718. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For easy
selection of recombinant virus, the plasmid contains the
beta-galactosidase gene from E. coli under control of a weak
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Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. The inserted
genes are flanked on both sides by viral sequences for cell-
mediated homologous recombination with wild-type viral DNA
to generate viable virus that expresses the cloned
polynucleotide.
Other baculovirus vectors are used in place of the
vector above, such as pAc373, pVL941 and pAcIMl, as one
skilled in the art would readily appreciate, as long as the
io construct provides appropriately located signals for
transcription, translation, secretion and the like,
including a signal peptide and an in-frame AUG as required.
Such vectors are described, for instance, in Luckow, et al.,
Virology 170:31-39.
The cDNA sequence encoding the mature hOB-BP2h
polypeptide in the deposited or other clone, lacking the AUG
initiation codon and the naturally associated nucleotide
binding site, is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene. Non-
limiting examples include 5' and 3' primers having
nucleotides corresponding or complementary to a portion of
the coding sequence of a hOB-BP2h polypeptide, e.g., as
presented in at least one of SEQ ID N0:1 and SEQ ID N0:2,
according to known method steps.
The amplified fragment is isolated from a 1~ agarose
gel using a commercially available kit (e. g., "Geneclean,"
BIO 101 Inc., La Jolla, CA). The fragment then is then
digested with the appropriate restriction enzyme and again
is purified on a 1~ agarose gel. This fragment is
3o designated herein "F1".
The plasmid is digested with the corresponding
restriction enzymes and optionally, can be dephosphorylated
using calf intestinal phosphatase, using routine procedures
known in the art. The DNA is then isolated from a 1~
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agarose gel using a commercially available kit ("Geneclean"
BIO 101 Inc., La Jolla, CA). This vector DNA is designated
herein "V1".
Fragment F1 and the dephosphorylated plasmid V1 are
ligated together with T4 DNA ligase. E. coli HB101 or other
suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning
Systems, La Jolla, CA) cells are transformed with the
ligation mixture and spread on culture plates. Bacteria are
identified that contain the plasmid with the human hOB-BP2h
1o gene using the PCR method, in which one of the primers that
is used to amplify the gene and the second primer is from
well within the vector so that only those bacterial colonies
containing the hOB-BP2h gene fragment will show
amplification of the DNA. The sequence of the cloned
fragment is confirmed by DNA sequencing. This plasmid is
designated herein pBac hOB-BP2h .
Five ~,g of the plasmid pBachOB-BP2h is co-transfected
with 1.0 ~.g of a commercially available linearized
baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen,
2o San Diego, CA), using the lipofection method described by
Felgner, et al., Proc. Natl. Acad. Sci. USA 84:7413-7417
(1987). 1 ~g of BaculoGoldTM virus DNA and 5 ~g of the
plasmid pBac hOB-BP2h are mixed in a sterile well of a
microtiter plate containing 50 ~1 of serum-free Grace's
medium (Life Technologies, Inc., Rockville, 1~).
Afterwards, 10 ~1 Lipofectin plus 90 ~1 Grace's medium are
added, mixed and incubated for 15 minutes at room
temperature. Then the transfection mixture is added drop-
wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm
3o tissue culture plate with 1 ml Grace's medium without serum.
The plate is rocked back and forth to mix the newly added
solution. The plate is then incubated for 5 hours at 27°C.
After 5 hours the transfection solution is removed from the
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plate and 1 ml of Grace's insect medium supplemented with
10~ fetal calf serum is added. The plate is put back into
an incubator and cultivation is continued at 27°C for four
days.
After four days the supernatant is collected and a
plaque assay is performed, according to known methods. An
agarose gel with "Blue Gal" (Life Technologies, Inc.,
Rockville, Nm) is used to allow easy identification and
isolation of gal-expressing clones, which produce blue-
1o stained plaques. (A detailed description of a "plaque
assay" of this type can also be found in the user's guide
for insect cell culture and baculovirology distributed by
Life Technologies, Inc., Rockville, MD, page 9-10). After
appropriate incubation, blue stained plaques are picked with
a micropipettor tip (e. g., Eppendorf). The agar containing
the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 ~1 of Grace's medium and
the suspension containing the recombinant baculovirus is
used to infect Sf9 cells seeded in 35 mm dishes. Four days
later the supernatants of these culture dishes are harvested
and then they are stored at 4°C. The recombinant virus is
called V-hOB-BP2h.
To verify the expression of the hOB-BP2h gene, Sf9
cells are grown in Grace's medium supplemented with 10~
heat-inactivated FBS. The cells are infected with the
recombinant baculovirus V-hOB-BP2h at a multiplicity of
infection ("MOI") of about 2. Six hours later the medium is
removed and is replaced with SF900 II medium minus
methionine and cysteine (available, e.g., from Life
3o Technologies, Inc., Rockville, MD). If radiolabeled
polypeptides are desired, 42 hours later, 5 mCi of 35S-
methionine and 5 mCi 35S-cysteine (available from Amersham)
are added. The cells are further incubated for 16 hours and
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then they are harvested by centrifugation. The polypeptides
in the supernatant as well as the intracellular polypeptides
are analyzed by SDS-PAGE followed by autoradiography (if
radiolabeled). Microsequencing of the amino acid sequence
of the amino terminus of purified polypeptide can be used to
determine the amino terminal sequence of the mature
polypeptide and thus the cleavage point and length of the
secretory signal peptide.
1o Example 3: Cloning and Expression of h08-BP2h in Mammnalian
Cells
A typical mammalian expression vector contains at least
one promoter element, which mediates the initiation of
transcription of mRNA, the polypeptide coding sequence, and
signals required for the termination of transcription and
polyadenylation of the transcript. Additional elements
include enhancers, Kozak sequences and intervening sequences
flanked by donor and acceptor sites for RNA splicing.
Highly efficient transcription is achieved with the early
2o and late promoters from SV40, the long terminal repeats
(LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early promoter of the cytomegalovirus (CMV). However,
cellular elements can also be used (e. g., the human actin
promoter). Suitable expression vectors for use in
practicing the present invention include, for example,
vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or
pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-),
pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL
and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152),
3o pSV2dhfr (ATCC 37146) and pBCI2MI (ATCC 67109). Mammalian
host cells that could be used include human Hela 293, H9 and
Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and
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CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster
ovary (CHO) cells.
Alternatively, the gene is expressed in stable cell
lines that contain the gene integrated into a chromosome.
The co-transfection with a selectable marker such as dhfr,
gpt, neomycin, or hygromycin allows the identification and
isolation of the transfected cells.
The transfected gene can also be amplified to express
large amounts of the encoded polypeptide. The DHFR
(dihydrofolate reductase) marker is useful to develop cell
lines that carry several hundred or even several thousand
copies of the gene of interest. Another useful selection
marker is the enzyme glutamine synthase (GS) (Murphy, et
al., Biochem. J. 227:277-279 (1991); Bebbington, et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells
with the highest resistance are selected. These cell lines
contain the amplified genes) integrated into a chromosome.
Chinese hamster ovary (CHO) and NSO cells are often used for
2o the production of polypeptides.
The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al.,
Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the
CMV-enhancer (Boshart, et al., Cell 41:521-530 (1985)).
Multiple cloning sites, e.g., with the restriction enzyme
cleavage sites BamHI, Xbal and Asp718, facilitate the
cloning of the gene of interest. The vectors contain in
addition the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene.
Example 3(a)s Cloning and Expression in COS Cells
The expression plasmid, phOB-BP2h HA, is made by
cloning a cDNA encoding hOB-BP2h into the expression vector
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pcDNAI/Amp or pcDNAIII (which can be obtained from
Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (1) an E.
coli origin of replication effective for propagation in E.
coli and other prokaryotic cells; (2) an ampicillin
resistance gene for selection of plasmid-containing
prokaryotic cells; (3) an SV40 origin of replication for
propagation in eucaryotic cells; (4) a CMV promoter, a
polylinker, an SV40 intron; (5) several codons encoding a
hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) or HIS tag (see, e.g, Ausubel, supra) followed
by a termination codon and polyadenylation signal arranged
so that a cDNA can be conveniently placed under expression
control of the CMV promoter and operably linked to the SV40
intron and the polyadenylation signal by means of
restriction sites in the polylinker. The HA tag corresponds
to an epitope derived from the influenza hemagglutinin
polypeptide described by Wilson, et al., Cell 37:767-778
(1984). The fusion of the HA tag to the target polypeptide
2o allows easy detection and recovery of the recombinant
polypeptide with an antibody that recognizes the HA epitope.
pcDNAIII contains, in addition, the selectable neomycin
marker.
A DNA fragment encoding the hOB-BP2h is cloned into the
polylinker region of the vector so that recombinant
polypeptide expression is directed by the CMV promoter. The
plasmid construction strategy is as follows. The hOB-BP2h
cDNA of the deposited clone is amplified using primers that
contain convenient restriction sites, much as described
3o above for construction of vectors for expression of hOB-BP2h
in E. coli. Non-limiting examples of suitable primers
include those based on the coding sequences presented in at
least one of SEQ ID N0:1 and SEQ ID N0:2, as they encode
hOB-BP2h polypeptides as described herein.
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The PCR amplified DNA fragment and the vector,
pcDNAI/Amp, are digested with suitable restriction enzymes)
and then ligated. The ligation mixture is transformed into
E. coli strain SURE (available from Stratagene Cloning
Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037),
and the transformed culture is plated on ampicillin media
plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or
other means for the presence of the hOB-BP2h-encoding
fragment.
For expression of recombinant hOB-BP2h, COS cells are
transfected with an expression vector, as described above,
using DEAE-DEXTR.AN, as described, for instance, in Sambrook,
et al., Molecular Cloning: a Laboratory Manual, Cold Spring
Laboratory Press, Cold Spring Harbor, New York (1989).
Cells are incubated under conditions for expression of hOB-
BP2h by the vector.
Expression of the hOB-BP2h-HA fusion polypeptide is
detected by radiolabeling and immunoprecipitation, using
methods described in, for example Harlow, et al.,
Antibodies: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (1988). To
this end, two days after transfection, the cells are labeled
by incubation in media containing 35S-cysteine for 8 hours.
The cells and the media are collected, and the cells are
washed and lysed with detergent-containing RIPA buffer: 150
mM NaCl, 1~ NP-40, 0.1~ SDS, 0.5~ DOC, 50 mM TRIS, pH 7.5,
as described by Wilson, et al. cited above. Proteins are
3o precipitated from the cell lysate and from the culture media
using an HA-specific monoclonal antibody. The precipitated
polypeptides then are analyzed by SDS-PAGE and
autoradiography. An expression product of the expected size
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is seen in the cell lysate, which is not seen in negative
controls.
Exsu~le 3(b): Cloning and Expression in CHO Calls
The vector pC4 is used for the expression of hOB-BP2h
polypeptide. Plasmid pC4 is a derivative of the plasmid
pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains
the mouse DHFR gene under control of the SV40 early
promoter. Chinese hamster ovary- or other cells lacking
l0 dihydrofolate activity that are transfected with these
plasmids are selected by growing the cells in a selective
medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The
amplification of the DHFR genes in cells resistant to
methotrexate (MTX) has been well documented (see, e.g., F.
W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L.
Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143
(1990); and M. J. Page and M. A. Sydenham, Biotechnology
9:64-68 (1991)). Cells grown in increasing concentrations
of MTX develop resistance to the drug by overproducing the
target enzyme, DHFR, as a result of amplification of the
DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in
the art that this approach is used to develop cell lines
carrying more than 1,000 copies of the amplified gene(s).
Subsequently, when the methotrexate is withdrawn, cell lines
are obtained which contain the amplified gene integrated
into one or more chromosomes) of the host cell.
Plasmid pC4 contains for expressing the gene of
3o interest the strong promoter of the long terminal repeat
(LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec.
Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from
the enhancer of the immediate early gene of human
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cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530
(1985)). Downstream of the promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow
integration of the genes. Behind these cloning sites the
plasmid contains the 3' intron and polyadenylation site of
the rat preproinsulin gene. Other high efficiency promoters
can also be used for the expression, e.g., the human b-actin
promoter, the SV40 early or late promoters or the long
terminal repeats from other retroviruses, e.g., HIV and
l0 HTLVI. Clontech's Tet-Off and Tet-On gene expression
systems and similar systems are used to express the hOB-BP2h
in a regulated way in mammalian cells (M. Gossen, and H.
Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
For the polyadenylation of the mRNA other signals, e.g.,
from the human growth hormone or globin genes is used as
well. Stable cell lines carrying a gene of interest
integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, 6418
or hygromycin. It is advantageous to use more than one
2o selectable marker in the beginning, e.g., G418 plus
methotrexate.
The plasmid pC4 is digested with restriction enzymes
and then dephosphorylated using calf intestinal phosphatase
by procedures known in the art. The vector is then isolated
from a 1~ agarose gel.
The DNA sequence encoding the complete hOB-BP2h
polypeptide is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene. Non-
limiting examples include 5' and 3' primers having
3o nucleotides corresponding or complementary to a portion of
the coding sequence of a hOB-BP2h, e.g., as presented in at
least one of SEQ ID N0:1 and SEQ ID N0:2, according to known
method steps.
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The amplified fragment is digested with suitable
endonucleases and then purified again on a 1~ agarose gel.
The isolated fragment and the dephosphorylated vector are
then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue
cells are then transformed and bacteria are identified that
contain the fragment inserted into plasmid pC4 using, for
instance, restriction enzyme analysis.
Chinese hamster ovary (CHO) cells lacking an active
DHFR gene are used for transfection. 5 ~.g of the expression
1o plasmid pC4 is cotransfected with 0.5 wg of the plasmid
pSV2-neo using lipofectin. The plasmid pSV2neo contains a
dominant selectable marker, the neo gene from Tn5 encoding
an enzyme that confers resistance to a group of antibiotics
including 6418. The cells are seeded in alpha minus MEM
supplemented with 400 ~g/ml 6418. After 2 days, the cells
are trypsinized and seeded in hybridoma cloning plates
(Greiner, Germany) in alpha minus MEM supplemented with 10,
25, or 50 ng/ml of methotrexate plus 400 ~g/ml 6418. After
about 10-14 days single clones are trypsinized and then
2o seeded in 6-well petri dishes or 10 ml flasks using
different concentrations of methotrexate (50 nM, 100 nM, 200
nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new
6-well plates containing even higher concentrations of
methotrexate (1 E1M, 2 ).,~,M, 5 E.4M, 10 ),.t,M, 20 EtM) . The same
procedure is repeated until clones are obtained which grow
at a concentration of 100 - 200 ~.~,M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE
and Western blot or by reverse phase HPLC analysis.
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E~cam~le 4: Cloning and Construction of hOB-BP2h-ECD
Ioglobulia Fusion Proteins (hOB-BP2h-ECD-Fc)
A. PREPARATION OF hOB-BP2h-ECD-FC FUSION PROTEINS
The extracellular domain (ECD) portion of hOB-BP2h is
prepared as a fusion protein coupled to an immunoglobulin
constant region (Fc), resulting in a hOB-BP2h-ECD-Fc
1o polypeptide. The immunoglobulin constant region may contain
genetic modifications including those which reduce or
eliminate effector activity inherent in the immunoglobulin
structure. (See, e.g., PCT Publication No. W088/07089,
published September 22, 1988). Briefly, PCR overlap
extension is applied to join DNA encoding the ECD portion of
a hOB-BP2h polypeptide to DNA encoding the hinge, CH2 and
CH3 regions of human IgGl. This is accomplished as
described in the following subsections.
B. PREPARATION OF GENE FUSIONS
A DNA fragment corresponding to the DNA sequences
encoding the hOB-BP2h-ECD or a portion thereof is prepared
by polymerase chain reaction (PCR). A cDNA encoding hOB-
BP2h can serve as the template for amplifying the hOB-BP2h-
ECD or portion thereof. PCR amplification is performed to
generate a DNA fragment which encodes the hOB-BP2h-ECD
(amino acid residues 1-360 of SEQ ID N0:3 or amino acid
residues 1-487 of SEQ ID N0:4) or a portion thereof (i.e.,
3o amino acid residues 41-360 of SEQ ID N0:3 or amino acid
residues 41-487 of SEQ ID N0:4).
In a second PCR reaction, a set of primers is designed
to amplify the IgG constant region (i.e., the hinge, CH2,
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and CH3, domains) such that the reverse primer has a unique
restriction site and the sequence of the forward primer has
a 5' terminus that is complementary to the 5' terminal
region of the reverse primer used in the hOB-BP2h-ECD
amplification and allowing the open reading frame in the
hOB-BP2h-ECD encoding nucleotide sequence to continue
throughout the length of the IgG nucleotide sequence. The
sequence of human IgG1 is obtained through Genbank
(accession: HUMIGCC4; Takahashi et. al (vo1.29, 671-679,
l0 1982). This is compiled into exons and a region upstream of
the natural hinge region is chosen as the fusion site. The
5' primer is designed to include an overlap for the hOB-
BP2h-ECD amplicon and yet amplify DNA encoding the Fc region
of human IgGl. The 3' primer is designed to amplify the DNA
molecule encoding the Fc region of human IgG1 while
incorporating both a translation stop codon and a
restriction site to facilitate cloning into the
amplification product. The target DNA in this reaction is
human genomic DNA encoding IgG heavy chain (Ellison et al.,
1982, Nuc. Acids. Res. 10:4071-4079) and is amplified using
Human Lymph Node QUICK-Clones cDNA purchased from Clontech
(cat# 7164-1) as template.
PCR reactions are prepared in 100 ~1 final volume
composed of Pfu polymerase and buffer (Stratagene)
containing primers (1 ~M each), dNTPs (200 ~M each), and 1ng
of template DNA.
The complete hOB-BP2h-ECD-Fc fusion segment is prepared
by performing another PCR reaction. The purified products
of the two PCR reactions above is mixed, denatured (95°C, 1
3o minute) and then renatured (54°C, 30 seconds) to allow
complementary ends of the two fragments to anneal. The
strands then are filled in using dNTPs and Taq polymerase
and the entire fragment is amplified using the forward PCR
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primer of the first PCR reaction and the reverse PCR primer
of the second PCR reaction. For convenience of cloning into
the expression vector, the resulting fragment is then
cleaved with restriction enzymes which recognize the unique
sites incorporated into the forward PCR primer of the first
PCR reaction and the reverse PCR primer of the second PCR
reaction. The digested fragment is gel purified and then
ligated into an expression vector, such as pIGl, that has
also been treated with the same restriction enzymes and calf
intestinal alkaline phosphatase (CIAP). The ligation
reaction is used to transform DHSa and recombinant plasmids
are identified. Plasmid DNA from isolated transformants is
prepared and the insert contained within the recombinant
plasmid is sequenced to confirm the correctness of the hOB-
BP2h-ECD-Fc fusion construct.
C. ISOLATION OF STABLE CLONES
Cell lines including, but not limited to, 293T cells
2o are transformed with recombinant plasmids containing the
hOB-BP2h-ECD-Fc fusion construct. For example, 293T cells
are grown and a transient transfection utilizing
lipofectamine(GIBCO-BRL) is performed. Characterization of
the supernatant revealing a protein of the size one would
expect for either a monomer or a dimer of the hOB-BP2h-ECD-
Fc, can confirm the integrity of the construct. The
expression of the protein can also be confirmed by a Western
utilizing an antibody to human IgGl.
To produce cell lines stably expressing the hOB-BP2h-
3o ECD-Fc fusion protein, a cell line such as, but not limited
to, the Syrian hamster cell line AV12-RGT18 is transfected
with the hOB-BP2h-ECD-Fc fusion protein construct by a
transfection method such as, but not limited to, the calcium
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chloride precipitation method (Promega). Two days after the
transfection the cells are washed and then trypsinized. The
cells are collected and resuspended in 10 ml of the
appropriate media. The transfected cells are plated onto
gridded Falcon 3025 plates at 1/10, 1/50, and 1/250 in a
final volume of 35 ml. The media may contain methotrexate at
250 nM concentration. pIG1 contains a copy of the DHFR gene
and when amplified will convey methotrexate resistance on
the transfected cells. After two to five days, colonies are
1o identified in the 1/50 and 1/250 dilution platings,
transferred to microtiter plates, and grown under selection.
The ability of these clones to produce the hOB-BP2h-ECD-Fc
protein is examined in serum free media. Single clones
producing hOB-BP2h-ECD-Fc protein are isolated and grown up
in 80 roller bottles. The media is collected and the hOB
BP2h-ECD-Fc fusion protein is isolated as described below.
Those skilled in the art are aware of various
considerations which influence the choice of expression
vector into which the hOB-BP2h-ECD-Fc fusion segment is
2o cloned, such as the identity of the host organism and the
presence of elements necessary for achieving desired
transcriptional and translational control. For example, if
transient expression is desired, the hOB-BP2h-ECD-Fc fusion
segment generated supra is cloned into the expression vector
pcDNA-1 (Invitrogen). Alternatively, stable expression of
the fusion protein is achieved by cloning the hOB-BP2h-ECD-
Fc fusion segment into the expression vector pcDNA-3
(Invitrogen). Alternatively, hOB-BP2h-ECD-Fc fusion
proteins is generated using an expression vector such as the
3o CD5-IgG1 vector (described by Aruffo et al., (1990), Cell,
61:1303-1313), which already contains the IgG constant
region. According to this method, the DNA fragment encoding
the hOB-BP2h-ECD is generated in a PCR reaction so that the
open reading frame encoding the hOB-BP2h-ECD is continuous
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and in frame with that encoding the IgG constant region.
For example, the extracellular domains (including signal
peptides) of hOB-BP2h-ECD are PCR amplified. Each forward
primer above contains a restriction site and each reverse
primer above contains a restriction site. After
amplification using the hOB-BP2h-ECD cDNA as a template, the
resulting PCR fragment is cloned into the CD5-IgG vector
(Aruffo et al., (1990), Cell). The resulting vector is
transiently transfected into COS cells and conditioned media
1o is generated. Immunoprecipitation (IP) of the conditioned
media with protein A and analysis by SDS PAGE reveals
whether the desired protein is expressed. To improve
expression of the human HOB-BP2H-ECD-Fc fusion, primers are
designed which amplify the extracellular domain of hOB-BP2H
(without the signal peptide) and this fragment is coligated
with sequences encoding other signal-peptides such as that
from mouse hOB-BP2h into the CD5-IgG vector. After
amplification, restriction enzyme digestion, and subcloning,
the resulting construct is transiently expressed in COS
cells. IP and SDS-PAGE analysis of the resulting
conditioned media will show whether expression of the human
h0B-BP2h-ECD-Fc fusion was successful. An alternative
method for enhancing the expression of immunoglobulin fusion
proteins, involves insertion of the hOB-BP2h-ECD (not
including the signal peptide) into the CD5-IgG1 vector in
such a manner so that the CD5 signal peptide is fused to the
mature hOB-BP2h-ECD. Such a signal peptide fusion has been
shown to improve expression of immunoglobulin fusion
proteins.
C. PREPARATION OF MODIFIED CH2 DOMAINS
The nucleotide sequence of the hOB-BP2h-ECD-Fc gene
fusions described supra, is modified to replace cysteine
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residues in the hinge region with serine residues and/or
amino acids within the CH2 domain which are believed to be
required for IgG binding to Fc receptors and complement
activation.
Modification of the CH2 domain to replace amino acids
thought to be involved in binding to Fc receptor is
accomplished as follows. The plasmid construct generated
supra, provides the template for modifications of the hOB-
BP2h-ECD-IgC'y1 CH2 domain. This template is PCR amplified
1o using the forward PCR primer described in the first PCR
reaction supra and a reverse primer designed such that it is
homologous to the 5' terminal portion of the CH2 domain of
IgG1 except for five nucleotide substitutions designed to
change amino acids 234, 235, and 237 (Canfield, S.M. and
Morrison, S.L., (1991), J. Exp. Med. 173:1483-1491) from Leu
to Ala, Leu to Glu, and Gly to Ala, respectively.
Amplification with these PCR primers will yield a DNA
fragment consisting of a modified portion of the CH2 domain.
In a second PCR reaction, the template is PCR amplified with
the reverse primer used in the second PCR reaction supra,
and a forward primer which is designed such that it is
complementary to the Ig portion of the molecule and contains
the five complementary nucleotide changes necessary for the
CH2 amino acid replacements. PCR amplification with these
primers will yield a fragment consisting of the modified
portion of the CH2 domain, an intron, the CH3 domain, and 3'
additional sequences. The complete hOB-BP2h-ECD-IgC~y1
segment consisting of a modified CH2 domain is prepared by
an additional PCR reaction. The purified products of the
3o two PCR reactions above are mixed, denatured (95°C, 1
minute) and then renatured (54°C, 30 seconds) to allow
complementary ends of the two fragments to anneal. The
strands are filled in using dNTP and Taq polymerase and the
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entire fragment is amplified using the forward PCR primer of
the first PCR reaction and the reverse PCR primer of the
second PCR reaction. For convenience of cloning into the
expression vector, the resulting fragment is then cleaved
with restriction enzymes which recognize those sites
specific to the forward PCR primer of the first PCR reaction
and the reverse PCR primer of the second PCR raction. This
digested fragment is then cloned into an expression vector
that was also treated with these restriction enzymes.
Sequence analysis is used to confirm structure and the
construct is used to transfect COS cells to test transient
expression. hIgG ELISA is used to measure/confirm transient
expression levels approximately equal to 100ng protein/ml
cell supernatant for the construct. CHO is one but not the
only example of a cell line that is transfected for
permanent expression of the fusion proteins.
Example 5: Isolation of a high-producing hOB-BP2h-ECD-Fc
clone from AY12 RGT18 traasfectaats
The vector pIG1 encodes resistance to methotrexate. In
addition, the vector is designed to contain a gene encoding
a fluorescent protein, GFP, on the same transcript and
immediately 3' to an inserted hOB-BP2h-ECD-Fc cDNA. In this
case, high level expression of GFP would require a high
level of expression of the hOB-BP2h-ECD-Fc mRNA. Thus,
highly fluorescent clones would have a greater probability
of producing high levels of hOB-BP2h-ECD-Fc. After
transfecting AV12 RGT18 cells with either pIG1-hOB-BP2h-ECD
3o and pIG1-h0B-BP2h-ECD-Fc cells resistant to 250 nM
methotrexate are selected and pooled. The pool of resistant
clones is subjected to fluorescence assisted cell sorting
(FRCS), and cells having fluorescence values in the top 5~
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of the population are sorted into a pool. This highly
fluorescent pool is subjected to three successive sorting
cycles. Pools and individual clones from the second and
third cycles are analyzed for hOB-BP2h-ECD production by
SDS-PAGE. Pools or clones expressing the hOB-BP2h-ECD
proteins at the highest level judged from Coomassie staining
are used for scale-up and purification of the expressed
protein.
Example 6: Purification of hOB-BP2h-ECD-Fc Fusion Proteins
io from AY12 media
AV12 cells transformed with a vector containing a cDNA
insert encoding a hOB-BP2h-ECD-Fc fusion protein are grown
in culture bottles until confluent. Media is collected,
concentrated approximately 20-fold, and clarified by
centrifugation. The media concentrate is pumped onto a Ni
loaded iminodiacetic acid column. The column is washed with
100 mM sodium phosphate, 100 mM sodium chloride buffer (pH
7.5). Bound protein is eluted with a pH gradient from pH
7.5 to 4.25.
Fractions containing the hOB-BP2h-ECD-Fc fusion protein
is pooled, diluted 1:1 with 50 mM sodium phosphate (pH 5.6)
and pumped onto a cation exchange column (TSK-SP 5PW). The
column is washed with 50 mM sodium phosphate (pH 5.6) and
bound protein eluted with a gradient from 0 to 0.5 M sodium
chloride. Fractions containing hOB-BP2h-ECD-Fc fusion are
pooled and dialyzed into phosphate buffered saline (pH 7.5).
The identity of the protein is confirmed by digesting
the protein with trypsin and analyzing the resulting
peptides by mass spectroscopy and tandem MS/MS analysis.
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Exa~le 7: Tissue Distribution of hOB-BP2h mRNA Expression
Northern blot. analysis is carried out to examine hOB-
BP2h gene expression in human tissues, using methods
described by, among others, Sambrook, et al., cited above.
A cDNA probe containing the entire nucleotide sequence of a
hOB-BP2h polypeptide (e.g., SEQ ID N0:1 or SEQ ID N0:2) is
labeled with 32P using the RediprimeTM DNA labeling system
(Amersham Life Science), according to the manufacturer's
instructions. After labeling, the probe is purified using a
CHROMA SPIN-100TM column (Clontech Laboratories, Inc.),
according to the manufacturer's protocol number PT1200-1.
The purified and labeled probe is used to examine various
human tissues for hOB-BP2h mRNA.
Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are
obtained from Clontech and are examined with the labeled
probe using ExpressHyb hybridization solution (Clontech)
according to manufacturer's protocol number PT1190-1.
Following hybridization and washing, the blots are mounted
2o and exposed to film at -70°C overnight, and films developed
according to standard procedures. The results show hOB-BP2h
polypeptides to be selectively expressed in at least one of
hemic, immune, and digestive system and other tissues.
EXAMPLE 8: Directed Mutagenesis of hOB-BP2h polypeptides to
provide DNA encoding specified substitutions, insertions or
deletions of SEQ ID N0:3, 4, or 5 Using the Polymerase Chain
Reaction
The polymerase chain reaction (PCR) is used for the
enzymatic amplification and direct sequencing of small
quantities of nucleic acids (see, e.g., Ausubel, supra,
section 15) to provide specified substitutions, insertions
or deletions in DNA encoding a hOB-BP2h polypeptide of the
present inventions (e.g., SEQ ID N0:1, SEQ ID N0:2, or any
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sequence described herein),
as presented herein, to provide
a hOB-BP2h polypeptide sequence
of interest including at
least one substitution, insertion
or deletion selected from
the group consisting of 3P, L, 8P, 9L, 11W, 15L, 16Q, 17E,
4
18K, 19P, 20V, 21Y, 22E, 23L, 24Q, 27K, 30T, 32Q, 37V, 38L,
47W, 48R, 495, 51Y, 525, 54P, 56L, 58V, 70A, 71E, 72V, 77N,
78P, 79D, 81R, 83K, 84P, 85E, 87Q, 91R, 93L, 96V, 97Q, 99K,
1045, 1066, 1098, 111E, 113T, 1146, 115S, 1248, 125D, 127K,
1295, 130Y, 131Q, 132Q, 133N, 134K, 135L, 136N, 138E, 141V,
101435, 143I, 144F, 144E, 145T, 210N, and 252A of SEQ ID N0:3,
or 3P, 4L, 8P, 9L, 11W, 15L, 16Q, 17E, 18K, 19P, 20V, 21Y,
22E, 23L, 24Q, 27K, 30T, 32Q, 37V, 38L, 47W, 48R, 49S, 51Y,
52S, 54P, 56L, 58V, 70A, 71E, 72V, 77N, 78P, 79D, 81R, 83K,
84P, 85E, 87Q, 91R, 93L, 96V, 97Q, 99K, 104S, 1066, 1098,
15111E, 113T, 1146, 1155, 1248, 125D, 127K, 1295, 130Y, 131Q,
132Q, 133N, 134K, 135L, 136N, 138E, 141A, 143T, 143I, 144Q,
144E, 145K, 152D, 194D, 3865, 387L, 389Q, 4058, 408F, 4098,
4118, 417C, 4198, 421E, 423K, 424P, 4326, 435K, 437N, 4386,
450I, 458D, 460K, 461V, 4625, 464K, 468I, 469Y, 4715, and
20476V of SEQ ID N0:4.
This technology is used as a quick and efficient method
for introducing any desired
sequence change into the DNA
of
interest.
Unit 8.5 of Ausubel, supra,
contains two basic
25protocols for introducing base
changes into specific DNA
sequences. Basic Protocol 1, as presented in the first
section 8.5 of Ausubel, supra (entirely incorporated herein
by reference), describes the incorporation of a restriction
site and Basic Protocol 2, as presented below and in the
3osecond section of Unit 8.5 of Ausubel, supra, details the
generation of specific point mutations (all of the following
references in this example are
to sections of Ausubel et
al., eds., Current Protocols in Molecular Biology, Wiley
Interscience, New York (1987- 1999)). An alternate protocol
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describes generating point mutations by sequential PCR
steps. Although the general procedure is the same in all
three protocols, there are differences in the design of the
synthetic oligonucleotide primers and in the subsequent
cloning and analyses of the amplified fragments.
The PCR procedure described here can rapidly,
efficiently, and reproducibly introduce any desired change
into a DNA fragment. It is similar to the oligonucleotide-
directed mutagenesis method described in UNIT 8.1, but does
1o not require the preparation of a uracil-substituted DNA
template.
The main disadvantage of PCR-generated mutagenesis is
related to the fidelity of the Taq DNA polymerase. The
mutation frequency for Taq DNA polymerase was initially
estimated to be as high as 1/5000 per cycle (Saiki et al.,
1988). This means that the entire amplified fragment must be
sequenced to be sure that there are no Taq-derived
mutations. To reduce the amount of sequencing required, it
is best to introduce the mutation by amplifying as small a
2o fragment as possible. With rapid and reproducible methods of
double-stranded DNA sequencing (UNIT 7.4), the entire
amplified fragment can usually be sequenced from a single
primer. If the fragment is somewhat longer, it is best to
subclone the fragment into an M13-derived vector, so that
both forward and reverse primers is used to sequence the
amplified fragment.
If there are no convenient restriction sites flanking
the fragment of interest, the utility of this method is
somewhat reduced. Many researchers prefer the mutagenesis
procedure in UNIT 8.1 to avoid excessive sequencing.
A full discussion of critical parameters for PCR
amplification is found in UNIT 15.1.
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Anticipated Results
Each of the procedures presented here has a 100
efficiency rate. All or substantially all of the cloned,
amplified fragments will contain the mutation corresponding
to the synthesized oligonucleotide.
Literature Cited
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J.,
Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A.
1988. Primer-directed enzymatic amplification of DNA with a
1o thermostable DNA polymerase. Science 239:487-491.
BASIC PROTOCOL (2): INTRODUCTION OF POINT MUTATIONS BY PCR
In this protocol, synthetic oligonucleotides are
designed to incorporate a point mutation at one end of an
amplified fragment. Following PCR, the amplified fragments
are made blunt-ended by treatment with Klenow fragment.
These fragments are then ligated and subcloned into a vector
to facilitate sequence analysis. This procedure is
summarized in Figure 8.5.2 of Ausubel, supra.
Materials
DNA sample to be mutagenized
Klenow fragment of E. coli DNA polymerase I (UNIT 3.5 of
Ausubel, supra)
Appropriate restriction endonuclease (Table 8.5.1)
Additional reagents and equipment for synthesis and
purification of oligonucleotides (UNITS 2.11 & 2.12),
phosphorylation of oligonucleotides (UNIT 3.10),
electrophoresis of DNA on nondenaturing agarose and low
gelling/melting agarose gels (UNITS 2.5A & 2.6), restriction
endonuclease digestion (UNIT 3.1), ligation of DNA fragments
(UNIT 3.16), transformation of E. coli (UNIT 1.8), plasmid
DNA miniprep (UNIT 1.6), and DNA sequence analysis (UNIT
7.4).
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Prepare the template DNA aad oligonucleotide primers
Prepare template DNA (see Basic Protocol 1, steps 1 and
2). Synthesize (UNIT 2.11) and purify (UNIT 2.12) the
oligonucleotide primers (primers 3 and 4 in Fig. 8.5.2B).
The oligonucleotide primers must be homologous to the
template DNA for more than 15 bases. No four-base "clamp"
sequence is added to these primers. The primer sequences
are based on a DNA encoding the hOB-BP2h polypeptide
sequence of interest including at least one substitution,
1o insertion or deletion selected from the group consisting of
3P, 4L, 8P, 9L, 11W, 15L, 16Q, 17E, 18K, 19P, 20V, 21Y, 22E,
23L, 24Q, 27K, 30T, 32Q, 37V, 38L, 47W, 48R, 495, 51Y, 525,
54P, 56L, 58V, 70A, 71E, 72V, 77N, 78P, 79D, 81R, 83K, 84P,
85E, 87Q, 91R, 93L, 96V, 97Q, 99K, 1045, 1066, 1098, 111E,
113T, 1146, 1155, 1248, 125D, 127K, 1295, 130Y, 131Q, 132Q,
133N, 134K, 135L, 136N, 138E, 141V, 1435, 143I, 144F, 144E,
145T, 210N, and 252A of SEQ ID N0:3, or 3P, 4L, 8P, 9L,
11W, 15L, 16Q, 17E, 18K, 19P, 20V, 21Y, 22E, 23L, 24Q, 27K,
30T, 32Q, 37V, 38L, 47W, 48R, 49S, 51Y, 525, 54P, 56L, 58V,
70A, 71E, 72V, 77N, 78P, 79D, 81R, 83K, 84P, 85E, 87Q, 91R,
93L, 96V, 97Q, 99K, 104S, 1066, 1098, 111E, 113T, 1146,
1155, 1248, 125D, 127K, 1295, 130Y, 131Q, 132Q, 133N, 134K,
135L, 136N, 138E, 141A, 143T, 143I, 144Q, 144E, 145K, 152D,
194D, 3865, 387L, 389Q, 4058, 408F, 4098, 4118, 417C, 4198,
421E, 423K, 424P, 4326, 435K, 437N, 4386, 450I, 458D, 460K,
461V, 4625, 464K, 468I, 469Y, 4715, and 476V of SEQ ID N0:4.
Phosphorylate the 5' end of the oligonucleotides
(UNIT 3.10). This step is necessary because the 5' end of
the oligonucleotide will be used directly in cloning.
3o Amplify DNA and prepare blunt-end fragments
Amplify the template DNA (see Basic Protocol 1, steps 5
and 6). After the final extension step, add 5 U Klenow
fragment to the reaction mix and incubate 15 min at 30°C.
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During PCR, the Taq polymerase adds an extra nontemplated
nucleotide to the 3' end of the fragment. The 3'-5'
exonuclease activity of the Klenow fragment is required to
make the ends flush and suitable for blunt-end cloning (UNIT
3.5). Analyze and process the reaction mix (see Basic
Protocol 1, steps 7 and 8). Digest half the amplified
fragments with the restriction endonucleases for the
flanking sequences (UNIT 3.1). Purify digested fragments on
a low gelling/melting agarose gel (UNIT 2.6).
Subclone the two amplified fragments into an
appropriately digested vector by blunt-end ligation (UNIT
3.16). Transform recombinant plasmid into E. coli (UNIT
1.8). Prepare DNA by plasmid miniprep (UNIT 1.6). Analyze
the amplified fragment portion of the plasmid DNA by DNA
sequencing to confirm the point mutation (UNIT 7.4). This
is critical because the Taq DNA polymerase can introduce
additional mutations into the fragment (see Critical
Parameters).
ALTERNATE PROTOCOL: INTRODUCTION OF A POINT MUTATION
BY SEQUENTIAL PCR STEPS
In this procedure, the two fragments encompassing the
mutation are annealed with each other and extended by
mutually primed synthesis; this fragment is then amplified
by a second PCR step, thereby avoiding the blunt-end
ligation required in Basic Protocol 2. This strategy is
outlined in Figure 8.5.3. For materials, see Basic Protocols
1 and 2 of Ausubel, supra.
Prepare template DNA (see Basic Protocol 1, steps 1 and
2). Synthesize (UNIT 2.11) and purify (UNIT 2.12) the
oligonucleotide primers (primers 5 and 6 in Fig. 8.5.3B) to
generate a hOB-BP2h polypeptide sequence of interest
including at least one substitution, insertion or deletion
selected from the group consisting of 3P, 4L, 8P, 9L, 11W,
15L, 16Q, 17E, 18K, 19P, 20V, 21Y, 22E, 23L, 24Q, 27K, 30T,
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32Q, 37V, 38L, 47w, 48R, 49s, 51Y, 525, 54P, 56L, 58v, 70A,
71E, 72V, 77N, 78P, 79D, 81R, 83K, 84P, 85E, 87Q, 91R, 93L,
96V, 97Q, 99K, 1045, 1066, 1098, 111E, 113T, 1146, 115 5,
1248, 125D, 127K, 1295, 130Y, 131Q, 132Q, 133N, 134K, 135L,
136N, 138E, 141V, 143S, 143I, 144F, 144E, 145T, 210N, and
252A of SEQ ID N0:3, or 3P, 4L, 8P, 9L, 11W, 15L, 16Q, 17E,
18K, 19P, 20V, 21Y, 22E, 23L, 24Q, 27K, 30T, 32Q, 37V, 38L,
47W, 48R, 49S, 51Y, 525, 54P, 56L, 58V, 70A, 71E, 72V, 77N,
78P, 79D, 81R, 83K, 84P, 85E, 87Q, 91R, 93L, 96V, 97Q, 99K,
1045, 1066, 1098, 111E, 113T, 1146, 1155, 1248, 125D, 127K,
129S, 130Y, 131Q, 132Q, 133N, 134K, 135L, 136N, 138E, 141A,
143T, 143I, 144Q, 144E, 145K, 152D, 194D, 386S, 387L, 389Q,
4058, 408F, 4098, 4118, 417C, 4198, 421E, 423K, 424P, 4326,
435K, 437N, 4386, 450I, 458D, 460K, 461V, 4625, 464K, 468I,
469Y, 4715, and 476V of SEQ ID N0:4.
The oligonucleotides must be homologous to the
template for 15 to 20 bases and must overlap with one
another by at least 10 bases. The 5' end does not have a
"clamp" sequence.
Amplify the template DNA and generate blunt-end
fragments (see Basic Protocol 2, steps 4 and 5). Puri fy
the
fragments by nondenaturing agarose gel electrophoresis (UNIT
2.5A). Resuspend in TE buffer at 1 ng/ul.
Carry out second PCR amplification. Combine the
following in a 500-ul microcentrifuge tube:
10 ul (10 ng) each amplified fragment
1 ul (500 ng) each flanking sequence primer (each 1 uM
final )
10 ul 10x amplification buffer
10 ul 2 mM 4dNTP mix
H20 to 99.5 ul
0.5 ul Taq DNA polymerase (5 U/ul).
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Overlay with 100 ul mineral oil. Carry out PCR for 20
to 25 cycles, using the conditions for introduction of
restriction endonuclease sites by PCR (see Basic Protocol 1,
step 6). Analyze and process the reaction mix (see Basic
Protocol 1, Ausubel, supra, steps 7 and 8).
Digest the DNA fragment with the appropriate
restriction endonuclease for the flanking sites (UNIT 3.1).
Purify the digested fragment on a low gelling/melting
agarose gel (UNIT 2.6). Subclone into an appropriately
1o digested vector. Transform recombinant plasmid into E. coli
(UNIT 1.8). Prepare DNA by plasmid miniprep (UNIT 1.6).
Analyze the amplified fragment portion of the plasmid DNA by
DNA sequencing (UNIT 7.4) to confirm the point mutation.
This is critical because the Taq DNA polymerase can
introduce additional mutations into the fragment (see
Critical Parameters)
It will be clear that the present invention is
practiced otherwise than as particularly described in the
foregoing description and examples.
2o Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
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SEQUENCE LISTING
<110> Eli Lilly and Company
<120> hOB-BP2h COMPOSITIONS,. METHODS AND USES THEREOF
<130> X12652
<140>
<141>
<160> 5
<170> PatentIn Ver. 2.1
<210> 1
<211> 1536
<212> DNA
<213> Homo sapiens
<400> 1
atgctactgc cactgctgct gtcctcgctg ctgggcgggt cccaggctat ggatgggaga 60
ttctggatac gagtgcagga gtcagtgatg gtgccggagg gcctgtgcat ctctgtgccc 120
tgctctttct cctacccccg acaggactgg acagggtcta ccccagctta tggctactgg 180
ttcaaagcag tgactgagac aaccaagggt gctcctgtgg ccacaaacca ccagagtcga 240
gaggtggaaa tgagcacccg gggccgattc cagctcactg gggatcccgc caaggggaac 300
tgctccttgg tgatcagaga cgcgcagatg caggatgagt cacagtactt ctttcgggtg 360
gagagaggaa gctatgtgag atataatttc atgaacgatg ggttctttct aaaagtaaca 420
gccctgactc agaagcctga tgtctacatc cccgagaccc tggagcccgg gcagccggtg 480
acggtcatct gtgtgtttaa ctgggccttt gaggaatgtc cacccccttc tttctcctgg 540
acgggggctg ccctctcctc ccaaggaacc aaaccaacga cctcccactt ctcagtgctc 600
agcttcacgc ccagacccca ggaccacgac accgacctca cctgccatgt ggacttctcc 660
agaaagggtg tgagcgcaca gaggaccgtc cgactccgtg tggcctatgc ccccagagac 720
cttgttatca gcatttcacg tgacaacacg ccagatcctc cagagaacct gagagtgatg 780
gtttcccaag caaacaggac agtcctggaa aaccttggga acggcacgtc tctcccagta 840
ctggagggcc aaagcctgtg cctggtctgt gtcacacaca gcagcccccc agccaggctg 900
agctggaccc agaggggaca ggttctgagc ccctcccagc cctcagaccc cggggtcctg 960
gagctgcctc gggttcaagt ggagcacgaa ggagagttca cctgccacgc tcggcaccca 1020
ctgggctccc agcacgtctc tctcagcctc tccgtgcact ataagaaggg actcatctca 1080
acggcattct ccaatggagc gtttctggga atcggcatca cggctcttct tttcctctgc 1140
ctggccctga tcatcatgaa gattctaccg aagagacgga ctcagacaga aaccccgagg 1200
cccaggttct cccggcacag cacgatcctg gattacatca atgtggtccc gacggctggc 1260
cccctggctc agaagcggaa tcagaaagcc acaccaaaca gtcctcggac ccctcttcca 1320
ccaggtgctc cctccccaga atcaaagaag aaccagaaaa agcagtatca gttgcccagt 1380
ttcccagaac ccaaatcatc cactcaagcc ccagaatccc aggagagcca agaggagctc 1440
cattatgcca cgctcaactt cccaggcgtc agacccaggc ctgaggcccg gatgcccaag 1500
ggcacccagg cggattatgc agaagtcaag ttccaa 1536
1
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<210> 2
<211> 1917
<212> DNA
<213> Homo sapiens
<400> 2
atgctactgc cactgctgct gtcctcgctg ctgggcgggt cccaggctat ggatgggaga 60
ttctggatac gagtgcagga gtcagtgatg gtgccggagg gcctgtgcat ctctgtgccc 120
tgctctttct cctacccccg acaagactgg acagggtcta ccccagctta tggctactgg 180
ttcaaagcag tgactgagac aaccaagggt gctcctgtgg ccacaaacca ccagagtcga 240
gaggtggaaa tgagcacccg gggccgattc cagctcactg gggatcccgc caaggggaac 300
tgctccttgg tgatcagaga cgcgcagatg caggatgagt cacagtactt ctttcgggtg 360
gagagaggaa gctatgtgag atataatttc atgaacgatg ggttctttct aaaagtaaca 420
gtgctcagct tcacgcccag accccaggac cacaacaccg acctcacctg ccatgtggac 480
ttctccagaa agggtgtgag cgcacagagg accgtccgac tccgtgtggc ctatgccccc 540
agagaccttg ttatcagcat ttcacgtgac aacacgccag ccctggagcc ccagccccag 600
ggaaatgtcc catacctgga agcccaaaaa ggccagttcc tgcggctcct ctgtgctgct 660
gacagccagc cccctgccac actgagctgg gtcctgcaga acagagtcct ctcctcgtcc 720
catccctggg gccctagacc cctggggctg gagctgcccg gggtgaaggc tggggattca 780
gggcgctaca cctgccgagc ggagaacagg cttggctccc agcagcgagc cctggacctc 840
tctgtgcagt atcctccaga gaacctgaga gtgatggttt cccaagcaaa caggacagtc 900
ctggaaaacc ttgggaacgg cacgtctctc ccagtactgg agggccaaag cctgtgcctg 960
gtctgtgtca cacacagcag ccccccagcc aggctgagct ggacccagag gggacaggtt 1020
ctgagcccct cccagccctc agaccccggg gtcctggagc tgcctcgggt tcaagtggag 1080
cacgaaggag agttcacctg ccacgctcgg cacccactgg gctcccagca cgtctctctc 1140
agcctctccg tgcactactc cccgaagctg ctgggcccct cctgctcctg ggaggctgag 1200
ggtctgcact gcagctgctc ctcccaggcc agcccggccc cctctctgcg ctggtggctt 1260
ggggaggagc tgctggaggg gaacagcagc caggactcct tcgaggtcac ccccagctca 1320
gccgggccct gggccaacag ctccctgagc ctccatggag ggctcagctc cggcctcagg 1380
ctccgctgtg aggcctggaa cgtccatggg gcccagagtg gatccatcct gcagctgcca 1440
gataagaagg gactcatctc aacggcattc tccaacggag cgtttctggg aatcggcatc 1500
acggctcttc ttttcctctg cctggccctg atcatcatga agattctacc gaagagacgg 1560
actcagacag aaaccccgag gcccaggttc tcccggcaca gcacgatcct ggattacatc 1620
aatgtggtcc cgacggctgg ccccctggct cagaagcgga atcagaaagc cacaccaaac 1680
agtcctcgga cccctcttcc accaggtgct ccctccccag aatcaaagaa gaaccagaaa 1740
aagcagtatc agttgcccag tttcccagaa cccaaatcat ccactcaagc cccagaatcc 1800
caggagagcc aagaggagct ccattatgcc acgctcaact tcccaggcgt cagacccagg 1860
cctgaggccc ggatgcccaa gggcacccag gcggattatg cagaagtcaa gttccaa 191?
<210> 3
<211> 512
<212> PRT
<213> Homo sapiens
<400> 3
Met Leu Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala
2
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1 5 10 15
Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro
20 25 30
Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln
35 40 45
Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val
50 55 60
Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg
65 70 75 80
Glu Val Glu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro
85 90 95
Ala Lys Gly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp
100 105 110
Glu Ser Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr
115 120 125
Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Ala Leu Thr Gln
130 135 140
Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val
145 150 155 160
Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys Pro Pro Pro
165 170 175
Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly Thr Lys Pro
180 185 190
Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr Pro Arg Pro Gln Asp
195 200 205
His Asp Thr Asp Leu Thr Cys His Val Asp Phe Ser Arg Lys Gly Val
210 215 220
Ser Ala Gln Arg Thr Val Arg Leu Arg Val Ala Tyr Ala Pro Arg Asp
225 230 235 240
Leu Val Ile Ser Ile Ser Arg Asp Asn Thr Pro Asp Pro Pro Glu Asn
245 250 255
Leu Arg Val Met Val Ser Gln Ala Asn Arg Thr Val Leu Glu Asn Leu
3
CA 02365040 2001-09-28
WO 00/59942 PCT/US00/06682
260 265 270
Gly Asn Gly Thr Ser Leu Pro Val Leu Glu Gly Gln Ser Leu Cys Leu
275 280 285
Val Cys Val Thr His Ser Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln
290 295 300
Arg Gly Gln Val Leu Ser Pro Ser Gln Pro Ser Asp Pro Gly Val Leu
305 310 315 320
Glu Leu Pro Arg Val Gln Val Glu His Glu Gly Glu Phe Thr Cys His
325 330 335
Ala Arg His Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser Val
340 345 350
His Tyr Lys Lys Gly Leu Ile Ser Thr Ala Phe Ser Asn Gly Ala Phe
355 360 365
Leu Gly Ile Gly Ile Thr Ala Leu Leu Phe Leu Cys Leu Ala Leu Ile
370 375 380
Ile Met Lys Ile Leu Pro Lys Arg Arg Thr Gln Thr Glu Thr Pro Arg
385 390 395 400
Pro Arg Phe Ser Arg His Ser Thr Ile Leu Asp Tyr Ile Asn Val Val
405 410 415
Pro Thr Ala Gly Pro Leu Ala Gln Lys Arg Asn Gln Lys Ala Thr Pro
420 425 430
Asn Ser Pro Arg Thr Pro Leu Pro Pro Gly Ala Pro Ser Pro Glu Ser
435 440 445
Lys Lys Asn Gln Lys Lys Gln Tyr Gln Leu Pro Ser Phe Pro Glu Pro
450 455 460
Lys Ser Ser Thr Gln Ala Pro Glu Ser Gln Glu Ser Gln Glu Glu Leu
465 470 475 480
His Tyr Ala Thr Leu Asn Phe Pro Gly Val Arg Pro Arg Pro Glu Ala
485 490 495
Arg Met Pro Lys Gly Thr Gln Ala Asp Tyr Ala Glu Val Lys Phe Gln
500 505 510
4
CA 02365040 2001-09-28
WO 00/59942 PCT/US00/06682
<210> 4
<211> 639
<212> PRT
<213> Homo sapiens
<400> 4
Met Leu Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala
1 5 10 15
Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro
20 25 30
Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln
35 40 45
Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val
50 55 60
Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg
65 70 75 80
Glu Val Glu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro
85 90 95
Ala Lys Gly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp
100 105 110
Glu Ser Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr
115 120 125
Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Val Leu Ser Phe
130 135 140
Thr Pro Arg Pro Gln Asp His Asn Thr Asp Leu Thr Cys His Val Asp
145 150 155 160
Phe Ser Arg Lys Gly Val Ser Ala Gln Arg Thr Val Arg Leu Arg Val
165 170 175
Ala Tyr Ala Pro Arg Asp Leu Val Ile Ser Ile Ser Arg Asp Asn Thr
180 185 190
Pro Ala Leu Glu Pro Gln Pro Gln Gly Asn Val Pro Tyr Leu Glu Ala
195 200 205
CA 02365040 2001-09-28
WO 00/59942 PCT/US00/06682
Gln Lys Gly Gln Phe Leu Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro
210 215 220
Pro Ala Thr Leu Ser Trp Val Leu Gln Asn Arg Val Leu Ser Ser Ser
225 230 235 240
His Pro Trp Gly Pro Arg Pro Leu Gly Leu Glu Leu Pro Gly Val Lys
245 250 255
Ala Gly Asp Ser Gly Arg Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly
260 265 270
Ser Gln Gln Arg Ala Leu Asp Leu Ser Val Gln Tyr Pro Pro Glu Asn
275 280 285
Leu Arg Val Met Val Ser Gln Ala Asn Arg Thr Val Leu Glu Asn Leu
290 295 300
Gly Asn Gly Thr Ser Leu Pro Val Leu Glu Gly Gln Ser Leu Cys Leu
305 310 315 320
Val Cys Val Thr His Ser Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln
325 330 335
Arg Gly Gln Val Leu Ser Pro Ser Gln Pro Ser Asp Pro Gly Val Leu
340 345 350
Glu Leu Pro Arg Val Gln Val Glu His Glu Gly Glu Phe Thr Cys His
355 360 365
Ala Arg His Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser Val
370 375 380
His Tyr Ser Pro Lys Leu Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu
385 390 395 400
Gly Leu His Cys Ser Cys Ser Ser Gln Ala Ser Pro Ala Pro Ser Leu
405 410 415
Arg Trp Trp Leu Gly Glu Glu Leu Leu Glu Gly Asn Ser Ser Gln Asp
420 425 430
Ser Phe Glu Val Thr Pro Ser Ser Ala Gly Pro Trp Ala Asn Ser Ser
435 440 445
Leu Ser Leu His Gly Gly Leu Ser Ser Gly Leu Arg Leu Arg Cys Glu
450 455 460
6
CA 02365040 2001-09-28
WO 00/59942 PCT/US00/06682
Ala Trp Asn Val His Gly Ala Gln Ser Gly Ser Ile Leu Gln Leu Pro
465 470 475 480
Asp Lys Lys Gly Leu Ile Ser Thr Ala Phe Ser Asn Gly Ala Phe Leu
485 490 495
Gly Ile Gly Ile Thr Ala Leu Leu Phe Leu Cys Leu Ala Leu Ile Ile
500 505 510
Met Lys Ile Leu Pro Lys Arg Arg Thr Gln Thr Glu Thr Pro Arg Pro
515 520 525
Arg Phe Ser Arg His Ser Thr Ile Leu Asp Tyr Ile Asn Val Val Pro
530 535 540
Thr Ala Gly Pro Leu Ala Gln Lys Arg Asn Gln Lys Ala Thr Pro Asn
545 550 555 560
Ser Pro Arg Thr Pro Leu Pro Pro Gly Ala Pro Ser Pro Glu Ser Lys
565 570 575
Lys Asn Gln Lys Lys Gln Tyr Gln Leu Pro Ser Phe Pro Glu Pro Lys
580 585 590
Ser Ser Thr Gln Ala Pro Glu Ser Gln Glu Ser Gln Glu Glu Leu His
595 600 605
Tyr Ala Thr Leu Asn Phe Pro Gly Val Arg Pro Arg Pro Glu Ala Arg
610 615 620
Met Pro Lys Gly Thr Gln Ala Asp Tyr Ala Glu Val Lys Phe Gln
625 630 635
<210> 5
<211> 58
<212> PRT
<213> Homo sapiens
<400> 5
Ala Leu Thr Gln Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro
1 5 10 15
Gly Gln Pro Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu
20 25 30
Cys Pro Pro Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln
35 40 45
7
CA 02365040 2001-09-28
WO 00/59942 PCT/US00/06682
Gly Thr Lys Pro Thr Thr Ser His Phe Ser
50 55
