U.S. patent application number 10/621263 was filed with the patent office on 2004-04-08 for human proteinase molecules.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Bandman, Olga, Baughn, Mariah R., Corley, Neil C., Guegler, Karl J..
Application Number | 20040067515 10/621263 |
Document ID | / |
Family ID | 21865378 |
Filed Date | 2004-04-08 |
United States Patent
Application |
20040067515 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
April 8, 2004 |
Human proteinase molecules
Abstract
The invention provides human proteinase molecules, the
polynucleotides which encode them, and methods for their use. The
invention also provides expression vectors, host cells, antibodies,
agonists, and antagonists. The invention further provides methods
for diagnosing or treating disorders associated with expression of
HPRM.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Corley, Neil C.; (Castro Valley, CA)
; Guegler, Karl J.; (Menlo Park, CA) ; Baughn,
Mariah R.; (Los Angeles, CA) |
Correspondence
Address: |
INCYTE CORPORATION
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
3160 Porter Drive
Palo Alto
CA
94304
|
Family ID: |
21865378 |
Appl. No.: |
10/621263 |
Filed: |
July 15, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10621263 |
Jul 15, 2003 |
|
|
|
09802633 |
Mar 8, 2001 |
|
|
|
6627605 |
|
|
|
|
09802633 |
Mar 8, 2001 |
|
|
|
09032523 |
Feb 27, 1998 |
|
|
|
6232454 |
|
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/226; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61P 37/02 20180101; A61K 38/00 20130101; C12N
9/6472 20130101; C07K 14/4705 20130101; C12N 9/6478 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/226; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 016/40; C12N 009/64 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1-3, b) a polypeptide comprising
a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3, c) a biologically active fragment of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-3, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-3.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:4-6.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-3.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, b) a
polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, c) a
polynucleotide complementary to a polynucleotide of a), d) a
polynucleotide complementary to a polynucleotide of b), and e) an
RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-3.
19. A method for treating a disease or condition associated with
decreased expression of functional HPRM, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional HPRM, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional HPRM, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A method for a diagnostic test for a condition or disease
associated with the expression of HPRM in a biological sample, the
method comprising: a) combining the biological sample with an
antibody of claim 11, under conditions suitable for the antibody to
bind the polypeptide and form an antibody:polypeptide complex, and
b) detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of HPRM in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, further comprising a label.
35. A method of diagnosing a condition or disease associated with
the expression of HPRM in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3, or
an immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibodies from the animal, and c)
screening the isolated antibodies with the polypeptide, thereby
identifying a polyclonal antibody which specifically binds to a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO: 1-3, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibody producing cells from the
animal, c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells, d)
culturing the hybridoma cells, and e) isolating from the culture
monoclonal antibody which specifically binds to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-3.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3 in a
sample, the method comprising: a) incubating the antibody of claim
11 with the sample under conditions to allow specific binding of
the antibody and the polypeptide, and b) detecting specific
binding, wherein specific binding indicates the presence of a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3 from
a sample, the method comprising: a) incubating the antibody of
claim 11 with the sample under conditions to allow specific binding
of the antibody and the polypeptide, and b) separating the antibody
from the sample and obtaining the purified polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
58. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
59. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
60. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:5.
61. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:6.
Description
[0001] This application is a DIVISIONAL of pending prior
application U.S. Ser. No. 09/802,633, filed on Mar. 8, 2001, which
in turn is a DIVISIONAL of prior application U.S. Ser. No.
09/032,523, filed on Feb. 27, 1998, now issued as U.S. Pat. No.
6,232,454 B1 on May 15, 2001, both entitled HUMAN PROTEINEASE
MOLECULES, both of which are hereby expressly incorporated by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human proteinase molecules and to the use of these
sequences in the diagnosis and treatment of cancer and immune
disorders.
BACKGROUND OF THE INVENTION
[0003] Proteolytic processing is an essential component of normal
cell growth, differentiation, remodeling, and homeostasis. The
cleavage of peptide bonds within cells is necessary for the
maturation of precursor proteins to their active form, the removal
of signal sequences from targeted proteins, the degradation of
incorrectly folded proteins, and the controlled turnover of
peptides within the cell. Proteases participate in apoptosis,
antigen presentation, inflammation, tissue remodeling during
embryonic development, wound healing, and normal growth. They are
necessary components of bacterial, parasitic, and viral invasion
and replication within a host. Four principal categories of
mammalian proteases have been identified based on active site
structure, mechanism of action, and overall three-dimensional
structure (Beynon and Bond (1994) Proteolytic Enzymes: A Practical
Approach, Oxford University Press, New York N.Y., pp. 1-5).
[0004] The serine proteases (SPs) are a large family of proteolytic
enzymes that include the digestive enzymes, trypsin and
chymotrypsin; components of the complement cascade and of the
blood-clotting cascade; and enzymes that control the degradation
and turnover of macromolecules of the extracellular matrix. SPs are
so named because of the presence of a serine residue found in the
active catalytic site for protein cleavage. The active site of all
SP is composed of a triad of residues including the aforementioned
serine, an aspartate, and a histidine residue. SPs have a wide
range of substrate specificities and can be subdivided into
subfamilies on the basis of these specificities. The main
sub-families are trypases which cleave after arginine or lysine;
aspases which cleave after aspartate; chymases which cleave after
phenylalanine or leucine; metases which cleavage after methionine;
and serases which cleave after serine.
[0005] The SPs are secretory proteins containing N-terminal signal
peptides which export the immature protein across the endoplasmic
reticulum prior to cleavage (von Heijne (1986) Nuc Acid Res
14:5683-5690). Differences in these signal sequences provide one
means of distinguishing individual SPs. Some SPs, particularly the
digestive enzymes, exist as inactive precursors or preproenzymes
and contain a leader or activation peptide on the C-terminal side
of the signal peptide. This activation peptide may be 2-12 amino
acids in length and extend from the cleavage site of the signal
peptide to the N-terminus of the active, mature protein. Cleavage
of this sequence activates the enzyme. This sequence varies in
different SPs according to the biochemical pathway and/or its
substrate (Zunino et al. (1990) J Immunol 144:2001-2009; Sayers et
al. (1994) J Immunol 152:2289-2297).
[0006] Cysteine proteases are involved in diverse cellular
processes ranging from the processing of precursor proteins to
intracellular degradation. Mammalian cysteine proteases include
lysosomal cathepsins and cytosolic calcium activated proteases,
calpains. Cysteine proteases are produced by monocytes, macrophages
and other cells of the immune system which migrate to sites of
inflammation and in their protective role secrete various molecules
to repair damaged tissue. These cells may overproduce the same
molecules and cause tissue destruction in certain disorders. In
autoimmune diseases such as rheumatoid arthritis, the secretion of
the cysteine protease, cathepsin C, degrades collagen, laminin,
elastin and other structural proteins found in the extracellular
matrix of bones. The cathepsin family of lysosomal proteases
includes the cysteine proteases; cathepsins B, H, K, L, O2, and S;
and the aspartyl proteases; cathepsins D and E. Various members of
this endosomal protease family are differentially expressed. Some,
such as cathepsin D, have a ubiquitous tissue distribution while
others, such as cathepsin L, are found only in monocytes,
macrophages, and other cells of the immune system.
[0007] Abnormal regulation and expression of cathepsins has been
implicated in various inflammatory disease states. In cells
isolated from inflamed synovia, the mRNA for stromelysin,
cytokines, TIMP-1, cathepsin, gelatinase, and other molecules is
preferentially expressed. Expression of cathepsins L and D is
elevated in synovial tissues from patients with rheumatoid
arthritis and osteoarthritis. Cathepsin L expression may also
contribute to the influx of mononuclear cells which exacerbate the
destruction of the rheumatoid synovium (Keyszer (1995) Arthritis
Rheum 38:976-984). The increased expression and differential
regulation of the cathepsins is linked to the metastatic potential
of a variety of cancers and may be of therapeutic and prognostic
interest (Chambers et al. (1993) Crit Rev Oncog 4:95-114).
[0008] Cysteine proteases are characterized by a catalytic domain
containing a triad of amino acid residues similar to that found in
serine proteases. A cysteine replaces the active serine residue.
Catalysis proceeds via a thiol ester intermediate and is
facilitated by the side chains of the adjacent histidine and
aspartate residues.
[0009] Aspartic proteases include bacterial penicillopepsin,
mammalian pepsin, renin, chymosin, cathepsins D and E, and certain
fungal proteases. The characteristic active site residues of
aspartic proteases are a pair of aspartic acid residues, such as
asp33 and asp213 in penicillopepsin. Aspartic proteases are also
called acid proteases because the optimum pH for activity is
between 2 and 3. In this pH range, only one of the aspartate
residues is ionized. A potent inhibitor of aspartic proteases is
the hexapeptide, pepstatin, which in the transition state resembles
a normal substrate of the enzyme.
[0010] Metalloproteases use zinc as an active site component and
are most notably represented in mammals by the exopeptidases,
carboxypeptidase A and B, and the matrix metalloproteases,
collagenase, gelatinase, and stromelysin. Carboxypeptidases A and B
are exopeptidases of similar structure and active sites.
Carboxypeptidase A, like chymotrypsin, prefers hydrophobic
C-terminal aromatic and aliphatic side chains, whereas
carboxypeptidase B is directed toward basic arginine and lysine
residues. The matrix-metalloproteases are secreted by connective
tissue cells and play an important role in the maintenance and
function of the basement membrane and extracellular matrix. A
naturally occurring inhibitor of metalloproteases, tissue inhibitor
of metalloproteases (TIMP), has been shown to prevent the invasion
of tumor cells through basement membrane, n vitro, indicating the
importance of these enzymes in cell invasion processes such as
tumor metastasis and the inflammatory response (Mignatti et al.
(1986) Cell 47:487-498).
[0011] Protease inhibitors play a major role in the regulation of
the activity and effect of proteases. They have been shown to
control pathogenesis in animal models of proteolytic disorders
(Murphy (1991) Agents Actions Suppl 35:69-76). In particular, low
levels of the cystatins, low molecular weight inhibitors of the
cysteine proteases, seem to be correlated with malignant
progression of tumors (Calkins et al. (1995) Biol Biochem Hoppe
Seyler 376:71-80). The balance between levels of cysteine proteases
and their inhibitors is also significant in the development of
disorders. Specifically, increases in cysteine protease levels,
when accompanied by reductions in inhibitor activity, are
correlated with increased malignant properties of tumor cells and
the pathology of arthritis and immunological diseases in
humans.
[0012] The discovery of new human proteinase molecules and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis and
treatment of cancer and immune disorders.
SUMMARY OF THE INVENTION
[0013] The invention features purified polypeptides, human
proteinase molecules, referred to collectively as "HPRM" and
individually as "HPRM-1", "HPRM-2", and "HPRM-3". In one
embodiment, the purified polypeptide, HPRM, comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, and SEQ ID NO:3, and fragments of SEQ ID NOs:1-3. The
invention includes a purified variant having at least 90% amino
acid identity to the amino acid sequences of SEQ ID NOs:1-3 or
fragments thereof.
[0014] The invention provides an isolated and purified
polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:1-3 and
fragments thereof. The invention also includes an isolated variant
having at least 90% sequence identity to the polynucleotide
encoding the polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs:1-3 and fragments
thereof.
[0015] The invention also provides an isolated polynucleotide
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and
fragments and complements of SEQ ID NOs:4-6. The invention includes
a variant having at least 90% sequence identity to the
polynucleotide selected from the group consisting of SEQ ID NOs:4-6
and complements and fragments thereof.
[0016] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 1-3 and fragments thereof. In
another aspect, the expression vector is contained within a host
cell. The invention still further provides a the method for using a
polynulceotide to produce a polypeptide comprising culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide encoding the polypeptide under
conditions for the expression of the polypeptide and recovering the
polypeptide from the host cell culture.
[0017] The invention yet still further provides a method for using
a polynucleotide to detect a nucleic acid encoding a polypeptide
having the amino acid sequence of SEQ ID NOs:1-3 in a sample
comprising hybridizing the polynucleotide or the complement thereof
to at least one nucleic acid in the sample, thereby forming a
hybridization complex and detecting the hybridization complex,
wherein the presence of the hybridization complex indicates the
expression of the nucleic acid in the sample. In one aspect, the
nucleic acids of the sample are amplified prior to
hybridization.
[0018] The invention additionally provides a method of using a
polynucleotide to screen a plurality of molecules to identify a
molecule which specifically binds the polynucleotide comprising
combining the polynucleotide with the plurality of molecules under
conditions to allow specific binding and detecting specific
binding, thereby identifying a molecule which specifically binds
the polynucleotide. In one aspect, the molecule is selected from
DNA molecules, RNA molecules, peptide nucleic acids, artificial
chromosome constructions, peptides, and proteins.
[0019] The method provides purified polypeptides comprising an
amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID
NO:3, and fragments thereof. In one aspect, a biologically active
fragment of the polypeptide is selected from residues D132-F144 of
SEQ ID NO: 1, residues C34-C415 of SEQ ID NO:2 and residues
V93-V104 of SEQ ID NO:3.
[0020] The invention also provides a method for using a polypeptide
to screen a plurality of molecules to identify a molecule which
specifically binds the polypeptide comprising combining the
polypeptide with the plurality of molecules under conditions to
allow specific binding and detecting specific binding, thereby
identifying a molecule which specifically binds the polypeptide. In
one aspect, the molecules are selected from agonists, antagonists,
antibodies, DNA molecules, RNA molecules, peptide nucleic acids,
immunoglobulins, inhibitors, drug compounds, peptides, and
pharmaceutical agents.
[0021] The invention further provides a method of using a
polypeptide to purify a molecule which specifically binds the
polypeptide from a sample comprising combining the polypeptide with
a sample under conditions to allow specific binding, recovering the
bound polypeptide, and separating the molecule from the
polypeptide, thereby obtaining the purified molecule.
[0022] The invention still further provides a method for using a
polypeptide to produce an antibody, comprising immunizing an animal
with the polypeptide under conditions to elicit an antibody
response and isolating antibodies which bind specifically to the
polypeptide.
[0023] The invention yet further provides a method for using a
polypeptide to purify an antibody which specifically binds the
polypeptide comprising combining the polypeptide with a plurality
of antibodies under conditions allow specific binding, recovering
the bound polypeptide, and separating the antibody from the
polypeptide, thereby obtaining antibody which specifically binds
the polypeptide. In one aspect, the antibodies are selected from
polyclonal antibodies, monoclonal antibodies, chimeric antibodies,
single chain antibodies; Fab fragments, Fv fragments, and
F(ab').sub.2 fragments.
[0024] The invention additionally provides a purified antibody
which specifically binds the polypeptide having the amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-3 and
fragments thereof.
[0025] The invention provides compositions comprising an isolated
polynucleotide encoding a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NOs:1-3 and fragments
thereof and reporter molecule or a purified polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NOs:1-3 and fragments thereof and a pharmaceutical carrier.
[0026] The invention also provides a method for treating or
preventing a cancer, the method comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of the polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NOs:1-3 and fragments
thereof.
[0027] The invention further provides a method for treating or
preventing an immune disorder, the method comprising administering
to a subject in need of such treatment an effective amount of an
antagonist of the polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NOs:1-3 and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID
NO:1) and nucleic acid sequence (SEQ ID NO:4) of HPRM-1. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering, San Bruno Calif.).
[0029] FIGS. 2A, 2B, 2C, 2D, and 2E show the amino acid sequence
(SEQ ID NO:2) and nucleic acid sequence (SEQ ID NO:5) of HPRM-2.
The alignment was produced using MACDNASIS PRO software.
[0030] FIGS. 3A, 3B, 3C, 3D, and 3E show the amino acid sequence
(SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:6) of HPRM-3.
The alignment was produced using MACDNASIS PRO software.
[0031] FIGS. 4A and 4B show the amino acid sequence alignments
between HPRM-1 (456885; SEQ ID NO:1), and a pig calpain I light
subunit (GI 164403; SEQ ID NO:7), produced using the multisequence
alignment program of LASERGENE software (DNASTAR, Madison
Wis.).
DESCRIPTION OF THE INVENTION
[0032] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0033] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
might be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0035] Definitions
[0036] "HPRM" refers to the amino acid sequences of purified HPRM
obtained from any species, particularly a mammalian species,
including bovine, ovine, porcine, murine, equine, and preferably
the human species, from any source, whether natural, synthetic,
semi-synthetic, or recombinant.
[0037] The term "agonist" refers to a molecule which, when bound to
HPRM, increases or prolongs the duration of the effect of HPRM.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of HPRM.
[0038] "Altered" nucleic acid sequences encoding HPRM include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polynucleotide the same HPRM or a
polypeptide with at least one functional characteristic of HPRM.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding HPRM, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding HPRM.
The encoded protein may also be "altered" and may contain
deletions, insertions, or substitutions of amino acid residues
which produce a silent change and result in a functionally
equivalent HPRM. Deliberate amino acid substitutions may be made on
the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the biological or immunological activity
of BPRM is retained. For example, negatively charged amino acids
may include aspartic acid and glutamic acid, positively charged
amino acids may include lysine and arginine, and amino acids with
uncharged polar head groups having similar hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine;
asparagine and glutamine; serine and threonine; and phenylalanine
and tyrosine.
[0039] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments", "immunogenic fragments",
or "antigenic fragments" refer to fragments of HPRM which are
preferably about 5 to about 15 amino acids in length and which
retain some biological activity or immunological activity of HPRM.
Where "amino acid sequence" is recited herein to refer to an amino
acid sequence of a naturally occurring protein molecule, "amino
acid sequence" and like terms are not meant to limit the amino acid
sequence to the complete native amino acid sequence associated with
the recited protein molecule.
[0040] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach and Dveksler (1995) PCR Primer, a
Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y., pp.
1-5).
[0041] The term "antagonist" refers to a molecule which, when bound
to HPRM, decreases the amount or the duration of the effect of the
biological or immunological activity of HPRM. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of HPRM.
[0042] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fa, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind HPRM polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0043] The term "antigenic determinant" refers to that fragment of
a molecule, an epitope, that makes contact with a particular
antibody. When a protein or a fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to antigenic
determinants, given regions or three-dimensional structures on the
protein. An antigenic determinant may compete with the intact
antigen, the immunogen used to elicit the immune response, for
binding to an antibody.
[0044] The term "biologically active" refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic HPRM, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0045] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides under permissive salt and
temperature conditions by base pairing. For example, the sequence
"A-G-T" binds to the complementary sequence "T-C-A". The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization. This
is of particular importance in amplification reactions, which
depend upon binding between nucleic acids strands, and in the
design and use of peptide nucleic acid (PNA) molecules. The
inhibition of hybridization of a completely complementary sequence
to the target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency.
[0046] A "composition comprising a given polynucleotide" or a
"composition comprising a given polypeptide" refer broadly to any
composition containing the given polynucleotide or polypeptide and
at least one other molecule. The other molecule specifically
encompasses labeling moeities, reporter molecules, and
pharmaceutical excipients and carriers.
[0047] "Consensus sequence" refers to a nucleic acid sequence which
has been resequenced to resolve uncalled bases, extended using
XL-PCR kit (Applied Biosystems (ABI), Foster City Calif.) in the 5'
and/or the 3' direction, and resequenced, or which has been
assembled from the overlapping sequences of more than one Incyte
Clone using a computer program for fragment assembly, such as the
GELVIEW Fragment Assembly system (Genetics Computer Group, Madison
Wis.). Some sequences have been both extended and assembled to
produce the consensus sequence.
[0048] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a polynucleotide encoding HPRM, by northern
analysis is indicative of the presence of nucleic acids encoding
HPRM in a sample, and thereby correlates with expression of the
transcript from the polynucleotide encoding HPRM.
[0049] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides.
[0050] The term "derivative" refers to the chemical modification of
HPRM, of a polynucleotide encoding HPRM, or of a polynucleotide
complementary to a polynucleotide encoding HPRM. Chemical
modifications of a polynucleotide can include, for example,
replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
polypeptide. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the natural
polypeptide.
[0051] The term "homology" refers to a degree of similarity or
"identity". "Percent identity" refers to the percentage of sequence
identity found in a comparison of two or more amino acid or nucleic
acid sequences. Percent identity can be determined electronically,
e.g., by using the multi-sequence alignment program of LASERGENE
software (DNASTAR). This program can create alignments between two
or more sequences according to selected method, e.g., the clustal
method (Higgins and Sharp (1988) Gene 73:237-244). The clustal
algorithm groups sequences into clusters by examining the distances
between all pairs. The clusters are aligned pairwise, and then, in
groups. The percentage identity between two amino acid sequences,
e.g., sequence A and sequence B, is calculated by dividing the
length of sequence A, minus the number of gap residues in sequence
A, minus the number of gap residues in sequence B, into the sum of
the residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no homology between the two amino acid
sequences are not included in determining percentage identity.
Percent identity between nucleic acid sequences can also be counted
or calculated by methods known in the art, e.g., the Jotun Hein
method (Hein (1990) Methods Enzymol 183:626-645).
[0052] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0053] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution or formed between one nucleic acid
sequence present in solution and another nucleic acid sequence
immobilized on a substrate.
[0054] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0055] The phrases "nucleic acid" or "nucleic acid sequence" refer
to an oligonucleotide, nucleotide, polynucleotide, or any fragment
thereof, to DNA or RNA of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent the sense or
the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers
to those nucleic acid sequences which are greater than about 60
nucleotides in length, and most preferably are at least about 100
nucleotides, at least about 1000 nucleotides, or at least about
10,000 nucleotides in length.
[0056] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the transcription of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0057] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. Oligonucleotide is equivalent to
the terms "amplimer", "primer", and "oligomer".
[0058] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA and RNA and stop transcript
elongation; they may be pegylated to extend their lifespan in the
cell (Nielsen et al. (1993) Anticancer Drug Des 8:53-63).
[0059] The term "sample" is used in its broadest sense. A
biological sample suspected of containing nucleic acids encoding
HPRM, or fragments thereof, or HPRM itself, may comprise a bodily
fluid; an extract from cell media, a cell, chromosome, organelle,
or membrane isolated from a cell; a cell; genomic DNA, RNA, or
cDNA, in solution or bound to a solid support; a tissue; a tissue
print; etc.
[0060] "Specific binding" refers to a specific interaction between
a nucleotide or protein and molecules with which it interacts.
These molecules include, but are not limited to, DNA molecules, RNA
molecules, peptide nucleic acids, artificial chromosome
constructions, peptides, proteins, agonists, antibodies,
antagonists, immunoglobulins, inhibitors, drug compounds, peptides,
and pharmaceutical agents. The interaction between the
polynucleotide or polypeptide and the bound molecule is dependent
upon the presence of a particular structure of the polynucleotide
or protein recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0061] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotide sequences and the
claimed polynucleotide sequences. Suitably stringent conditions can
be defined by, for example, the concentrations of salt or formamide
in the prehybridization and hybridization solutions, or by the
hybridization temperature, and are well known in the art. In
particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0062] The term "purified" refers to nucleic acid or amino acid
sequences that are removed from their natural environment or cell
culture media and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are associated.
[0063] "Specific binding" refers to a specific interaction between
a nucleotide or protein and molecules with which it interacts.
These molecules include, but are not limited to, DNA molecules, RNA
molecules, peptide nucleic acids, artificial chromosome
constructions, peptides, proteins, agonists, antibodies,
antagonists, immunoglobulins, inhibitors, drug compounds, peptides,
and pharmaceutical agents. The interaction between the
polynucleotide or polypeptide and the bound molecule is dependent
upon the presence of a particular structure of the polynucleotide
or protein recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0064] "Substrate" refers to any solid support including, but not
limited to, membranes, filters, chips, slides, wafers, fibers,
magnetic or nonmagnetic beads, gels, capillaries or other tubing,
plates, polymers, and microparticles with a variety of surface
forms including wells, trenches, pins, channels and pores to which
cells or their nucleic acids have been attached.
[0065] A "variant" of HPRM refers to a nucleotide or amino acid
sequence that is altered by one or more nucleotides or amino acids.
The variant may have "conservative" changes wherein the substituted
moiety has similar structural or chemical properties (e.g., a
purine is substituted for a purine or a leucine is replaced with an
isoleucine). Analogous minor variations may also include at least
one amino acid deletion or insertion, or both. Guidance in
determining which amino acid residues may be substituted, inserted,
or deleted without abolishing biological or immunological activity
may be found using computer programs well known in the art such as
LASERGENE software (DNASTAR).
[0066] The Invention
[0067] The invention is based on the discovery of new human
proteinase molecules (HPRM), the polynucleotides encoding HPRM and
the use of these compositions for the diagnosis and treatment of
cancer and immune disorders.
[0068] Nucleic acids encoding the HPRM-1 of the present invention
were first identified in Incyte Clone 456855 from the keratinocyte
cDNA library (KERANOT01) using a computer search for amino acid
sequence alignments. A consensus sequence, SEQ ID NO:4, was derived
from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 456855 (KERANOT01) and 3363138
(PROSBPT02).
[0069] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1 as shown in FIGS.
2A-2E. HPRM-1 is 248 amino acids in length and contains potential
phosphorylation sites for casein kinase II at T68, S73, S129, and
S237, and for protein kinase C at T123, T136, and S237, and for
tyrosine kinase at Y146. HPRM-1 also contains a potential EF-hand
calcium-binding domain between residues D132 and F144. As shown in
FIGS. 4A-4B, HPRM-1 has chemical and structural homology with the
calcium-binding, calpain I light subunit from pig (GI 164403; SEQ
ID NO:7). In particular, HPRM-1 and the pig calpain subunit share
65% homology. The pig calpain subunit shares the EF-hand
calcium-binding domain, and the potential phosphorylation sites
found at residues T68, T123, and Y146 in HPRM-1. A fragment of SEQ
ID NO:4 from about nucleotide 145 to about nucleotide 193 is useful
for hybridization. Northern analysis shows the expression of this
sequence in skin, neonatal keratinocytes, and hyperplastic prostate
cDNA libraries.
[0070] Nucleic acids encoding the HPRM-2 of the present invention
were first identified in Incyte Clone 947429 from the atrium tissue
cDNA library (RATRNOT02) using a computer search for amino acid
sequence alignments. A consensus sequence, SEQ ID NO:5, was derived
from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 947429 (RATRNOT02), 870803 and 877928
(LUNGAST01), 907964 (COLNNOTO9), and 2632243 (COLNTUT15).
[0071] In another embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, as
shown in FIGS. 2A-2E. HPRM-2 is 415 amino acids in length and has a
potential signal peptide sequence between residues M1 and Q23. A
potential N-glycosylation site is found at residue N355, and
potential phosphorylation sites are found for casein kinase II at
T64, S142, and T274, for protein kinase C at T60, T109, S164, S241,
and S357, and for tyrosine kinase at Y207. Cysteine residues,
representing potential intramolecular disulfide bridging sites are
found at residues C34, C59, C86, C107, C154, C181, C208, C231,
C297, C312, C364, and C415. HPRM-2 has chemical and structural
homology with mouse procollagen C-proteinase enhancer (GI
2589009;SEQ ID NO:8). In particular, HPRM-2 and mouse procollagen
C-proteinase enhancer share 42% homology, the phosphorylation sites
at T64, T109, and S357, and the twelve cysteine residues found in
HPRM-2. A fragment of SEQ ID NO:5 from about nucleotide 405 to
about nucleotide 513 is useful for hybridization. Northern analysis
shows the expression of this sequence in various libraries, at
least 45% of which are immortalized or cancerous and at least 31%
of which involve immune response. Of particular note is the
expression of HPRM in tumors of the testes, lung, heart, colon, and
bladder, and in inflammatory conditions including rheumatoid
arthritis, asthma, and Crohn's disease.
[0072] Nucleic acids encoding the HPRM-3 of the present invention
were first identified in Incyte Clone 1516165 from the pancreatic
tumor cDNA library (PANCTUT01) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:6, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 1516165 (PANCTUT01), 1360069 (LUNGNOT12),
794210 (OVARNOT03), and shotgun sequences SAWA02729, SAWA00677,
SAWA01399, and SAWA00459.
[0073] In another embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:3, as
shown in FIGS. 3A-3E. HPRM-3 is 349 amino acids in length and has a
potential signal peptide sequence from residue M1 to A17, a
potential N-glycosylation site at residue N90, and potential
phosphorylation sites for casein kinase II at S65, S168, T175,
S221, T293, and S333, and for protein kinase C at S31 and S65.
HPRM-3 also contains a potential eukaryotic aspartyl protease
active site signature sequence between residues V93 and V104, in
which D96 is the catalytic site. HPRM-3 has chemical and structural
homology with human cathepsin E precursor (GI 181194; SEQ ID NO:9).
In particular, HPRM-3 and the human cathepsin E precursor share 88%
homology. HPRM-3 is an apparent splice variant of the human
cathepsin E precursor in which the sequence of the latter molecule
between residues 1263 and E309 has been deleted. The fragment of
SEQ ID NO:6 from about nucleotide 807 to about nucleotide 857,
which encompasses this deletion, is useful for hybridization.
Northern analysis shows the expression of this sequence in various
libraries, at least 61% of which are immortalized or cancerous and
at least 32% of which involve immune response. Of particular note
is the expression of HPRM-3 in tumors of the ovaries, pancreas,
testes, and lung, and in inflammatory conditions including asthma,
lymphocytic thyroiditis, and inflamed adenoids.
[0074] The invention also encompasses HPRM variants. A preferred
HPRM variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the BPRM amino acid sequence, and which
contains at least one functional or structural characteristic of
HPRM.
[0075] The invention also encompasses polynucleotides which encode
HPRM. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:4, as
shown in FIGS. 1A-1C, which encodes an HPRM. In a further
embodiment, the invention encompasses the polynucleotide sequence
comprising the sequence of SEQ ID NO:5, as shown in FIGS. 2A-2E. In
a further embodiment, the invention encompasses the polynucleotide
sequence comprising the sequence of SEQ ID NO:6, as shown in FIGS.
3A-3E.
[0076] The invention also encompasses a variant of a polynucleotide
sequence encoding HPRM. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding HPRM. A particular aspect of the invention encompasses a
variant of SEQ ID NO:4 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:4. The invention
further encompasses a polynucleotide variant of SEQ ID NO:5 having
at least about 80%, more preferably at least about 90%, and most
preferably at least about 95% polynucleotide sequence identity to
SEQ ID NO:5. The invention further encompasses a polynucleotide
variant of SEQ ID NO:6 having at least about 80%, more preferably
at least about 90%, and most preferably at least about 95%
polynucleotide sequence identity to SEQ ID NO:6. Any one of the
polynucleotide variants described above can encode an amino acid
sequence which contains at least one functional or structural
characteristic of HPRM.
[0077] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding HPRM, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring HPRM, and all such variations are
to be considered as being specifically disclosed.
[0078] Although nucleotide sequences which encode HPRM and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HPRM under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HPRM or its derivatives
possessing a different codon usage. Codons may be selected to
increase the rate at which expression of the peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for altering the nucleotide sequence encoding HPRM
and its derivatives without altering the encoded amino acid
sequences include the production of RNA transcripts having more
desirable properties, such as a greater half-life, than transcripts
produced from the naturally occurring sequence.
[0079] The invention also encompasses production of DNA sequences
which encode HPRM and HPRM derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding HPRM or any fragment
thereof.
[0080] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NOs:4-6 or fragments thereof under various conditions of
stringency (Wahl and Berger (1987) Methods Enzymol 152:399-407;
Kimmel (1987) Methods Enzymol 152:507-511).
[0081] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq
polymerase, thermostable T7 polymerase (Amersham Pharmacia Biotech
(APB), Piscataway N.J.), or combinations of polymerases and
proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Rockville Md.).
Preferably, the process is automated with machines such as the
MICROLAB 2200 (Hamilton, Reno Nev.), DNA ENGINE thermal cycler (MJ
Research, Waltham Mass.) and the CATALYST and 373 and 377 PRISM DNA
sequencing systems (ABI).
[0082] The nucleic acid sequences encoding HPRM may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus (Sarkar (1993)
PCR Methods Applic 2:318-322). In particular, genomic DNA is first
amplified in the presence of a primer which is complementary to a
linker sequence within the vector and a primer specific to a region
of the nucleotide sequence. The amplified sequences are then
subjected to a second round of PCR with the same linker primer and
another specific primer internal to the first one. Products of each
round of PCR are transcribed with an appropriate RNA polymerase and
sequenced using reverse transcriptase.
[0083] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia et al.
(1988) Nucleic Acids Res 16:8186). The primers may be designed
using commercially available software such as OLIGO software
(Molecular Biology Insights, Cascade Colo.) or another appropriate
program to be about 22 to 30 nucleotides in length, to have a GC
content of about 50% or more, and to anneal to the target sequence
at temperatures of about 68.degree. C. to 72.degree. C. The method
uses several restriction enzymes to generate a fragment in the
known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template.
[0084] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom
et al (1991) PCR Methods Applic 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may be used to
place an engineered double-stranded sequence into an unknown
fragment of the DNA molecule before performing PCR. Other methods
which may be used to retrieve unknown sequences are known in the
art (Parker et al. (1991) Nucleic Acids Res 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERF NDER
libraries to walk genomic DNA (Clontech, Palo Alto Calif.). This
process avoids the need to screen libraries and is useful in
finding intron/exon junctions.
[0085] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0086] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR software, ABI),
and the entire process from loading of samples to computer analysis
and electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0087] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HPRM may be used in
recombinant DNA molecules to direct expression of HPRM, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode the same or a functionally equivalent
amino acid sequence may be produced, and these sequences may be
used to clone and express HPRM.
[0088] As will be understood by those of skill in the art, it may
be advantageous to produce HPRM-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0089] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HPRM-encoding sequences for a variety of reasons including,
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0090] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HPRM may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of HPRM activity, it may
be useful to encode a chimeric HPRM protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the HPRM
encoding sequence and the heterologous protein sequence, so that
HPRM may be cleaved and purified away from the heterologous
moiety.
[0091] In another embodiment, sequences encoding HPRM may be
synthesized, in whole or in part, using chemical methods well known
in the art (Caruthers et al (1980) Nucleic Acids Symp Ser (7)
215-223; Horn et al. (1980) Nucleic Acids Symp Ser (7) 225-232).
Alternatively, the protein itself maybe produced using chemical
methods to synthesize the amino acid sequence of HPRM, or a
fragment thereof. For example, peptide synthesis can be performed
using various solid-phase techniques (Roberge et al. (1995) Science
269:202-204). Automated synthesis may be achieved using the 431A
peptide synthesizer (ABI). Additionally, the amino acid sequence of
HPRM, or any part thereof, may be altered during direct synthesis
and/or combined with sequences from other proteins, or any part
thereof, to produce a variant polypeptide.
[0092] The peptide may be purified by preparative high performance
liquid chromatography (Chiez and Regnier (1990) Methods Enzymol
182:392-421). The composition of the synthetic peptides may be
confirmed by amino acid analysis or by sequencing (Creighton (1984)
Proteins, Structures and Molecular Properties, W H Freeman, New
York N.Y.).
[0093] In order to express a biologically active HPRM, the
nucleotide sequences encoding HPRM or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0094] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HPRM and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; Ausubel et al. (1995, and periodic
supplements) Current Protocols in Molecular Biology, John Wiley
& Sons, New York N.Y., ch. 9, 13, and 16.)
[0095] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HPRM. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0096] The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and 3'
untranslated regions, of the vector and polynucleotide sequences
encoding HPRM which interact with host cellular proteins to carry
out transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters,
e.g., hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,
La Jolla Calif.) or PSPORT1 plasmid (Life Technologies), may be
used. The baculovirus polyhedrin promoter may be used in insect
cells. Promoters or enhancers derived from the genomes of plant
cells (e.g., heat shock, RUBISCO, and storage protein genes) or
from plant viruses (e.g., viral promoters or leader sequences) may
be cloned into the vector. In mammalian cell systems, promoters
from mammalian genes or from mammalian viruses are preferable. If
it is necessary to generate a cell line that contains multiple
copies of the sequence encoding HPRM, vectors based on SV40 or EBV
may be used with an appropriate selectable marker.
[0097] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for HPRM. For example,
when large quantities of HPRM are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as BLUESCRIPT phagemid (Stratagene), in
which the sequence encoding HPRM may be ligated into the vector in
frame with sequences for the amino-terminal Met and the subsequent
7 residues of .beta.-galactosidase so that a hybrid protein is
produced, and pin vectors. (Van Heeke and Schuster (1989) J Biol
Chem 264:5503-5509). PGEX vectors (APB) may also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0098] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used (Ausubel, supra;
Grant et al. (1987) Methods Enzymol 153:516-544).
[0099] In cases where plant expression vectors are used, the
expression of sequences encoding HPRM may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu (1987) EMBO J
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi et
al. (1984) EMBO J 3:1671-1680; Broglie et al. (1984) Science
224:838-843; and Winter et al. (1991) Results Probl Cell Differ
17:85-105). These constructs can be introduced into plant cells by
direct DNA transformation or pathogen-mediated transfection. Such
techniques are described in a number of generally available reviews
(Hobbs or Murry (1992) In: Yearbook of Science and Technology,
McGraw Hill, New York N.Y.; pp. 191-196).
[0100] An insect system may also be used to express HPRM. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding HPRM may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of sequences
encoding HPRM will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein. The recombinant viruses may
then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which HPRM maybe expressed (Engelhard et al.
(1994) Proc Nat Acad Sci 91:3224-3227).
[0101] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HPRM may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing HPRM in
infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci
81:3655-3659). In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0102] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0103] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding HPRM. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding HPRM and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate for the particular cell
system used (Scharf et al. (1994) Results Probl Cell Differ
20:125-162).
[0104] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and W138), are available from the ATCC
(Bethesda Md.) and may be chosen to ensure the correct modification
and processing of the foreign protein.
[0105] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing HPRM can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0106] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes and adenine
phosphoribosyltransferase genes, which can be employed in tk or apr
cells, respectively (Wigler et al. (1977) Cell 11:223-232; Lowy et
al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For
example, dhfr confers resistance to methotrexate; npt confers
resistance to the aminoglycosides neomycin and G-418; and als or
pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Wigler et al. (1980) Proc Natl
Acad Sci 77:3567-3570; Colbere-Garapin et al. (1981) J Mol Biol
150:1-14; and Murry, supra). Additional selectable genes have been
described, e.g., trpB, which allows cells to utilize indole in
place of tryptophan, or hisD, which allows cells to utilize
histinol in place of histidine (Hartman and Mulligan (1988) Proc
Natl Acad Sci 85:8047-8051). Visible markers such as anthocyanins,
.beta. glucuronidase and its substrate GUS, luciferase and its
substrate luciferin may be used. Green fluorescent proteins (GFP;
Clontech) can also be used. These markers can be used not only to
identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system (Rhodes et al. (1995) Methods Mol Biol
55:121-131).
[0107] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding HPRM is inserted within a marker gene
sequence, transformed cells containing sequences encoding HPRM can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding HPRM under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0108] Alternatively, host cells which contain the nucleic acid
sequence encoding HPRM and express HPRM may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0109] The presence of polynucleotide sequences encoding HPRM can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
HPRM. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding HPRM
to detect transformants containing DNA or RNA encoding HPRM.
[0110] A variety of protocols for detecting and measuring the
expression of HPRM, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
HPRM is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art (Hampton et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St
Paul Minn., Section IV; Maddox et al. (1983) J Exp Med
158:1211-1216).
[0111] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HPRM include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HPRM, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits such as those
provided by APB. Reporter molecules or labels which may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0112] Host cells transformed with nucleotide sequences encoding
HPRM may be cultured under conditions for the expression and
recovery of the protein from cell culture. The protein produced by
a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode HPRM may be designed to
contain signal sequences which direct secretion of HPRM through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding HPRM to nucleotide sequences
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex., Seattle, Wash.).
The inclusion of cleavable linker sequences, such as those specific
for Factor XA (APB) or enterokinase (Invitrogen, Carlsbad Calif.),
between the purification domain and the HPRM encoding sequence may
be used to facilitate purification. One such expression vector
provides for expression of a fusion protein containing HPRM and a
nucleic acid encoding 6 histidine residues preceding a thioredoxin
or an enterokinase cleavage site. The histidine residues facilitate
purification on immobilized metal ion affinity chromatography
(Porath et al. (1992) Prot Exp Purif 3: 263-281). The enterokinase
cleavage site provides a means for purifying HPRM from the fusion
protein (Kroll et al. (1993) DNA Cell Biol 12:441-453).
[0113] Fragments of HPRM may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques (Creighton (1984) Protein: Structures and Molecular
Properties, pp. 55-60, W H Freeman, New York N.Y.). Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the 431A
peptide synthesizer (ABI). Various fragments of HPRM may be
synthesized separately and then combined to produce the full length
molecule.
[0114] Therapeutics
[0115] Chemical and structural homology exists among HPRM and the
calcium-binding, calpain I subunit from pig (GI 164403), a
procollagen-C proteinase enhancer protein from mouse (GI 2589009),
and an aspartic proteinase, cathepsin E, from human (GI 181194). In
addition, HPRM is expressed in cancer and the immune response.
Therefore, HPRM appears to play a role in cancer and immune
disorders.
[0116] Therefore, in one embodiment, an antagonist of HPRM may be
administered to a subject to treat or prevent a cancer. Such a
cancer may include, but is not limited to, adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds HPRM may be used directly as an
antagonist or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express
HPRM.
[0117] In another embodiment, a vector expressing the complement of
the polynucleotide encoding HPRM may be administered to a subject
to treat or prevent a cancer including, but not limited to, those
described above.
[0118] In another embodiment, an antagonist of HPRM may be
administered to a subject to treat or prevent an immune disorder.
Such an immune disorder may include, but is not limited to, AIDS,
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner
syndrome, and complications of cancer, hemodialysis, and
extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections; and trauma.
[0119] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding HPRM may be administered
to a subject to treat or prevent an immune disorder including, but
not limited to, those described above.
[0120] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment of the various disorders described above. Using this
approach, one may be able to achieve therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for
adverse side effects.
[0121] An antagonist of HPRM may be produced using methods which
are generally known in the art. In particular, purified HPRM may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind HPRM. Antibodies
to HPRM may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies, those which inhibit dimer formation, are
especially preferred for therapeutic use.
[0122] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with HPRM or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0123] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to HPRM have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of HPRM amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0124] Monoclonal antibodies to HPRM may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler et al. (1975)
Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods
81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and
Cole et al. (1984) Mol Cell Biol 62:109-120).
[0125] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used (Morrison
et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al.
(1984) Nature 312:604-608; and Takeda et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce HPRM-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random
combinatorial immunoglobulin libraries (Burton (1991) Proc Natl
Acad Sci 88:10134-10137).
[0126] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi et al. (1989) Proc
Natl Acad Sci 86: 3833-3837; Winter et al. (1991) Nature
349:293-299).
[0127] Antibody fragments which contain specific binding sites for
HPRM may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity (Huse et al. (1989) Science 246:1275-1281).
[0128] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between HPRM and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering HPRM epitopes
is preferred, but a competitive binding assay may also be employed
(Maddox, supra.).
[0129] In another embodiment of the invention, the polynucleotides
encoding HPRM, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding HPRM may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding HPRM. Thus, complementary molecules or
fragments may be used to modulate HPRM activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding HPRM.
[0130] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding HPRM (Sambrook, supra;
Ausubel, supra).
[0131] Genes encoding HPRM can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding HPRM. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0132] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5, or regulatory
regions of the gene encoding HPRM. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature (Gee et al. (1994) In: Huber
and Carr, Molecular and Immunologic Approaches, Futura Publishing,
Mt. Kisco N.Y., pp. 163-177). A complementary sequence or antisense
molecule may also be designed to block translation of mRNA by
preventing the transcript from binding to ribosomes.
[0133] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding HPRM.
[0134] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0135] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding HPRM. Such DNA sequences may be incorporated
into a wide variety of vectors with RNA polymerase promoters such
as T7 or SP6. Alternatively, these cDNA constructs that synthesize
complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or tissues.
[0136] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5 '
and/or 3' ends of the molecule, or the use of phosphorothioate or
2'O-methyl rather than phosphodiesterase linkages within the
backbone of the molecule. This concept is inherent in the
production of PNAs and can be extended in all of these molecules by
the inclusion of nontraditional bases such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine, cytidine, guanine, thynine, and uridine
which are not as easily recognized by endogenous endonucleases.
[0137] Many methods for introducing vectors into cells or tissues
are available for use in vivo, in vitro, and ex vivo. For ex vivo
therapy, vectors may be introduced into stem cells taken from the
patient and clonally propagated for autologous transplant back into
that same patient. Delivery by transfection, by liposome
injections, or by polycationic amino polymers may be achieved using
methods which are well known in the art (Goldman et al. (1997)
Nature Biotechnol 15:462-466).
[0138] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0139] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of HPRM, antibodies to HPRM, and mimetics,
agonists, antagonists, or inhibitors of HPRM. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0140] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0141] In addition to the active ingredients, these pharmaceutical
compositions may contain pharmaceutically-acceptable carriers
comprising excipients and auxiliaries which facilitate processing
of the active compounds into preparations which can be used
pharmaceutically. Further details on techniques for formulation and
administration may be found in the latest edition of Reminton's
Pharmaceutical Sciences (Maack Publishing, Easton Pa.).
[0142] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages for oral administration. Such carriers enable
the pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for ingestion by the patient.
[0143] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Excipients include
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, and sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate. Auxiliaries can be added, if desired.
[0144] Dragee cores may be used in conjunction with coatings, such
as concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets
or dragee coatings for product identification or to characterize
the quantity of active compound, i.e., dosage.
[0145] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
liquids, such as fatty oils, liquid, or liquid polyethylene glycol
with or without stabilizers.
[0146] Pharmaceutical formulations for parenteral administration
may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Lipophilic solvents or vehicles include fatty oils,
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate, triglycerides, or liposomes. Non-lipid polycationic amino
polymers may also be used for delivery. Optionally, the suspension
may also contain stabilizers or agents to increase the solubility
of the compounds and allow for the preparation of highly
concentrated solutions.
[0147] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0148] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0149] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0150] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of HPRM, such
labeling would include amount, frequency, and method of
administration.
[0151] Pharmaceutical compositions for use in the invention include
compositions wherein the active ingredients are contained in an
effective amount to achieve the intended purpose. The determination
of an effective dose is well within the capability of those skilled
in the art.
[0152] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, for example HPRM or fragments thereof,
antibodies of HPRM, and agonists, antagonists or inhibitors of
HPRM, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0155] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0156] Diagnostics
[0157] In another embodiment, antibodies which specifically bind
HPRM may be used for the diagnosis of disorders characterized by
expression of HPRM, or in assays to monitor patients being treated
with HPRM or agonists, antagonists, or inhibitors of HPRM.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for BPRM include methods which utilize the antibody and a label to
detect HPRM in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0158] A variety of protocols for measuring HPRM, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of HPRM expression. Normal or
standard values for HPRM expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to HPRM under conditions for
complex formation. The amount of standard complex formation may be
quantitated by various methods, preferably by photometric means.
Quantities of HPRM expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0159] In another embodiment of the invention, the polynucleotides
encoding HPRM may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HPRM may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of HPRM, and to
monitor regulation of HPRM levels during therapeutic
intervention.
[0160] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HPRM or closely related molecules may be used
to identify nucleic acid sequences which encode HPRM. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding HPRM, alleles, or related
sequences.
[0161] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the HPRM encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequences of SEQ ID NOs:4-6 or from genomic
sequences including promoters, enhancers, and introns of the HPRM
gene.
[0162] Means for producing specific hybridization probes for DNAs
encoding HPRM include the cloning of polynucleotide sequences
encoding HPRM or HPRM derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0163] Polynucleotide sequences encoding HPRM may be used for the
diagnosis of a disorder associated with expression of HPRM.
Examples of such a disorder include, but are not limited to,
cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
and uterus; and immune disorders such as AIDS, Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis,bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis,
hypereosinophilia, irritable bowel syndrome, lupus erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, ulcerative colitis, Werner syndrome, and
complications of cancer, hemodialysis, and extracorporeal
circulation; viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections; and trauma. The polynucleotide sequences
encoding HPRM may be used in Southern or northern analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and ELISA assays; and in microarrays utilizing
fluids or tissues from patients to detect altered HPRM expression.
Such qualitative or quantitative methods are well known in the
art.
[0164] In a particular aspect, the nucleotide sequences encoding
HPRM may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding HPRM may be labeled by standard methods and
added to a fluid or tissue sample from a patient under conditions
for the formation of hybridization complexes. After an incubation
period, the sample is washed and the signal is quantitated and
compared with a standard value. If the amount of signal in the
patient sample is significantly altered in comparison to a control
sample then the presence of altered levels of nucleotide sequences
encoding HPRM in the sample indicates the presence of the
associated disorder. Such assays may also be used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal
studies, in clinical trials, or to monitor the treatment of an
individual patient.
[0165] In order to provide a basis for the diagnosis of a disorder
associated with expression of HPRM, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding HPRM, under conditions for hybridization or amplification.
Standard hybridization may be quantified by comparing the values
obtained from normal subjects with values from an experiment in
which a known amount of a purified polynucleotide is used. Standard
values obtained in this manner may be compared with values obtained
from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of
a disorder.
[0166] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0167] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0168] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HPRM may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding HPRM, or a fragment of a
polynucleotide complementary to the polynucleotide encoding HPRM,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0169] Methods which may also be used to quantitate the expression
of HPRM include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves (Melby et al. (1993) J Immunol Methods
159:235-244; Duplaa et al. (1993) Anal Biochem 229-236). The speed
of quantitation of multiple samples may be accelerated by running
the assay in an ELISA format where the oligomer of interest is
presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid quantitation.
[0170] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0171] Microarrays may be prepared, used, and analyzed using
methods known in the art (Brennan, et al. (1995) U.S. Pat. No.
5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619;
Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon et
al. (1995) PCT application WO95/35505; Heller et al. (1997) Proc
Natl Acad Sci 94:2150-2155; and Heller et al. (1997) U.S. Pat. No.
5,605,662).
[0172] In another embodiment of the invention, nucleic acid
sequences encoding HPRM may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries (Price (1993) Blood Rev 7:127-134; Trask (1991) Trends
Genet 7:149-154).
[0173] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data (Heinz-Ulrich et aL. (1995) In: Meyers, Molecular Biology and
Biotechnology, VCH Publishers New York N.Y., pp. 965-968). Examples
of genetic map data can be found in various scientific journals or
at the Online Mendelian Inheritance in Man (OMIM) site. Correlation
between the location of the gene encoding HPRM on a physical
chromosomal map and a specific disorder, or a predisposition to a
specific disorder, may help define the region of DNA associated
with that disorder. The nucleotide sequences of the invention may
be used to detect differences in gene sequences among normal,
carrier, and affected individuals.
[0174] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., AT to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation (Gatti et al. (1988) Nature 336:577-580). The
nucleotide sequence of the subject invention may also be used to
detect differences in the chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
[0175] In another embodiment of the invention, HPRM, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between HPRM and the agent being tested may be
measured.
[0176] Another technique for drug screening provides for high
throughput screening of compounds having binding affinity to the
protein of interest (Geysen, et al. (1984) PCT application
WO84/03564). In this method, large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The test compounds are reacted with
HPRM, or fragments thereof, and washed. Bound HPRM is then detected
by methods well known in the art. Purified HPRM can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0177] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HPRM specifically compete with a test compound for binding
HPRM. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
HPRM.
[0178] In additional embodiments, the nucleotide sequences which
encode HPRM may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0179] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0180] I RATRNOT02 cDNA Library Construction
[0181] The right atrium tissue used for the RATRNOT02 library
construction was obtained from a 39 year old Caucasian male who
died of a gun shot wound. The frozen tissue was homogenized and
lysed using a POLYTRON homogenizer (Brinkmann Instruments, Westbury
N.Y.) in guanidinium isothiocyanate solution. The lysate was
centrifuged over a 5.7 M CsCI cushion using an SW28 rotor in a
L8-70M ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18
hours at 25,000 rpm at ambient temperature. The RNA was extracted
with phenol chloroform, pH 4.0, precipitated using 0.3 M sodium
acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water
and treated with DNase at 37.degree. C. RNA extraction and
precipitation were repeated as before. The MRNA was isolated with
the OLIGOTEX kit (Qiagen, Valencia Calif.) and used to construct
the cDNA library.
[0182] A 10 million clone cDNA library was constructed using three
micrograms of poly A.sup.+ mRNA and Not I/oligo d(T) primer. The
cDNAs were directionally inserted into Sal I/Not I sites of PSPORT1
plasmid (Life Technologies) and the plasmid was transformed into
competent DH5.alpha. cells or ELECTROMAX DH10B cells (Life
Technologies).
[0183] II Isolation and Sequencing of cDNA Clones
[0184] Plasmid DNA was released from the cells and purified using
the MINIPREP kit (Advanced Genetic Technologies, Gaithersburg Md.).
This kit consists of a 96-well block with reagents for 960
purifications. The recommended protocol was employed except for the
following changes: 1) the 96 wells were each filled with 1 ml of
sterile TERRIHC BROTH (BD Biosciences, Sparks Md.) containing
carbenicillin at 25 mg/l and glycerol at 0.4%; 2) the bacteria were
inoculated into the wells, cultured for 24 hours, and then lysed
with 60 .mu.l of lysis buffer; 3) the blocks were centrifuged in
the GS-6R rotor (Beckman Coulter) at 2900 rpm for 5 minutes, and
the contents of the block were added to the primary filter plate;
and 4) the optional step of adding isopropanol to TRIS buffer was
eliminated. After the last step in the protocol, samples were
transferred to a 96-well block for storage.
[0185] The cDNAs were prepared using a MICROLAB 2200 (Hamilton) in
combination with DNA ENGINE thermal cyclers (MJ Research) and
sequenced by the method of Sanger and Coulson (1975; J Mol Biol
94:441f) using 377 PRISM DNA sequencing systems (ABI); and the
reading frame was determined.
[0186] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0187] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool; Altschul (1993) J Mol Evol 36:290-300; Altschul et al.
(1990) J Mol Biol 215:403-410).
[0188] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties
(Smith et al. (1992) Protein Engineering 5:35-51). The sequences
disclosed in this application have lengths of at least 49
nucleotides and have no more than 12% uncalled bases (where N is
rpalces A, C, G, or T).
[0189] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0190] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0191] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., the
Block 2 Bioanalysis program (Incyte Genomics, Palo Alto Calif.).
This motif analysis program, based on sequence information
contained in the SwissProt Database and PROSITE, is a method of
determining the function of uncharacterized proteins translated
from genomic or cDNA sequences (Bairoch et al. (1997) Nucleic Acids
Res 25:217-221; Attwood et al. (1997) J Chem Inf Comput Sci
37:417-424). PROSITE maybe used to identify common functional or
structural domains in divergent proteins. The method is based on
weight matrices. Motifs identified by this method are then
calibrated against the SwissProt database in order to obtain a
measure of the chance distribution of the matches.
[0192] In another alternative, Hidden Markov models (HMMs) maybe
used to find protein domains, each defined by a data set of
proteins known to have a common biological function (Pearson and
Lipman (1988) Proc Natl Acad Sci 85:2444-2448; Smith and Waterman
(1981) J Mol Biol 147:195-197). HMMs were initially developed to
examine speech recognition patterns, but are now being used in a
biological context to analyze protein and nucleic acid sequences as
well as to model protein structure (Keogh et al. (1994) J Mol Biol
235:1501-1531; Collin et al. (1993) Protein Sci 2:305-314). HMMs
have a formal probabilistic basis and use position-specific scores
for amino acids or nucleotides. The algorithm continues to
incorporate information from newly identified sequences to increase
its motif analysis capabilities.
[0193] IV Northern Analysis
[0194] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook, supra, ch. 7; Ausubel, supra, ch. 4 and 16).
[0195] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Genomics). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or homologous.
[0196] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0197] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0198] The results of northern analysis are reported as a list of
libraries in which the transcript encoding HPRM occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0199] V Extension of HPRM Encoding Polynucleotides
[0200] The nucleic acid sequences of Incyte Clones 456855, 947429,
and 1516165 were used to design oligonucleotide primers for
extending partial nucleotide sequences to full length. For each
nucleic acid sequence, one primer was synthesized to initiate
extension of an antisense polynucleotide, and the other was
synthesized to initiate extension of a sense polynucleotide.
Primers were used to facilitate the extension of the known sequence
"outward" generating amplicons containing new unknown nucleotide
sequence for the region of interest. The initial primers were
designed from the cDNA using OLIGO software (Molecular Biology
Insights), or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the target sequence at temperatures of about
68.degree. C. to about 72.degree. C. Any stretch of nucleotides
which would result in hairpin structures and primer-primer
dimerizations was avoided.
[0201] Selected human cDNA libraries (Life Technologies) were used
to extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0202] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (ABI) and thoroughly mixing the
enzyme and reaction mix. PCR was performed using the DNA ENGINE
thermal cycler (MJ Research), beginning with 40 pmol of each primer
and the recommended concentrations of all other components of the
kit, with the following parameters: Step 1, 94.degree. C. for 1 min
(initial denaturation); Step 2, 65.degree. C. for 1 min; Step 3,
68.degree. C. for 6 min; Step 4, 94.degree. C. for 15 sec; Step 5,
65.degree. C. for 1 min; Step 6, 68.degree. C. for 7 min; Step 7,
repeat steps 4 through 6 for an additional 15 cycles; Step 8,
94.degree. C. for 15 sec; Step 9, 65.degree. C. for 1 min; Step 10,
68.degree. C. for 7:15 min; Step 11, repeat steps 8 through 10 for
an additional 12 cycles; Step 12, 72.degree. C. for 8 min; and Step
13, 4.degree. C. (and holding).
[0203] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK
(Qiagen), and trimmed of overhangs using Klenow enzyme to
facilitate religation and cloning.
[0204] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook, supra, Appendix A, p.
2). After incubation for one hour at 37.degree. C., the E. coli
mixture was plated on Luria Bertani (LB) agar (Sambrook, supra,
Appendix A, p. 1) containing carbenicillin (2.times.carb). The
following day, several colonies were randomly picked from each
plate and cultured in 150 .mu.l of liquid LB/2.times.carb medium
placed in an individual well of an appropriate
commercially-available sterile 96-well microtiter plate. The
following day, 5 .mu.l of each overnight culture was transferred
into a non-sterile 96-well plate and, after dilution 1:10 with
water, 5 .mu.l from each sample was transferred into a PCR
array.
[0205] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions: Step 1, 94.degree. C.
for 60 sec; Step 2, 94.degree. C. for 20 sec; Step 3, 55.degree. C.
for 30 sec; Step 4, 72.degree. C. for 90 sec; Step 5, repeat steps
2 through 4 for an additional 29 cycles; Step 6, 72.degree. C. for
180 sec; and Step 7, 4.degree. C. (and holding).
[0206] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0207] In like manner, the nucleotide sequences of SEQ ID NOs:4-6
are used to obtain 5'regulatory sequences using the procedure
above, oligonucleotides designed for 5'extension, and an
appropriate genomic library.
[0208] VI Labeling and Use of Individual Hybridization Probes
[0209] Hybridization probes derived from SEQ ID NO:4-6 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO software (Molecular Biology
Insights) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (APB), and T4
polynucleotide kinase (PerkinElmer Life Sciences, Boston Mass.).
The labeled oligonucleotides are purified using a SEPHADEX G-25
superfine resin column (APB). An aliquot containing 10.sup.7 counts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or
Pvu II (PerkinElmer Life Sciences).
[0210] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to NYTRAN PLUS membranes (Schleicher
& Schuell, Keene N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times.saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR film (Eastman Kodak,
Rochester N.Y.) is exposed to the blots for several hours,
hybridization patterns are compared.
[0211] VII Microarrays
[0212] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate
(Baldeschweiler, supra). An array analogous to a dot or slot blot
may also be used to arrange and link elements to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding
procedures. A typical array may be produced by hand or using
available methods and machines and contain any appropriate number
of elements. After hybridization, nonhybridized probes are removed
and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0213] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments for hybridization can be selected using software well
known in the art such as LASERGENE software (DNASTAR). Full-length
cDNAs, ESTs, or fragments thereof corresponding to one of the
nucleotide sequences of the present invention, or selected at
random from a cDNA library relevant to the present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA
is fixed to the slide using, e.g., UV cross-linking followed by
thermal and chemical treatments and subsequent drying (Schena et
al. (1995) Science 270:467-470; Shalon et al. (1996) Genome Res
6:639-645). Fluorescent probes are prepared and used for
hybridization to the elements on the substrate. The substrate is
analyzed by procedures described above.
[0214] VIII Complementary Polynucleotides
[0215] Sequences complementary to the HPRM-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring HPRM. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO software (Molecular Biology Insights) and the
coding sequence of HPRM. To inhibit transcription, a complementary
oligonucleotide is designed from the most unique 5'sequence and
used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent
ribosomal binding to the HPRM-encoding transcript.
[0216] IX Expression of HPRM
[0217] Expression of HPRM is accomplished by subcloning the cDNA
into an appropriate vector and transforming the vector into host
cells. This vector contains an appropriate promoter, e.g.,
B-galactosidase, upstream of the cloning site, operably associated
with the cDNA of interest (Sambrook, supra, pp. 404-433; Rosenberg
et al (1983) Methods Enzymol 101:123-138).
[0218] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of HPRM into bacterial growth media which can be used
directly in the following assay for activity.
[0219] X Demonstration of HPRM Activity
[0220] Protease activity of HPRM is measured by the hydrolysis of
appropriate synthetic peptide substrates conjugated with various
chromogenic molecules in which the degree of hydrolysis is
quantitated by spectrophotometric (or fluorometric) absorption of
the released chromophore (Beynon and Bond, supra pp.25-55). Peptide
substrates are designed according to the category of protease
activity as endopeptidase (serine, cysteine, aspartic proteases),
animopeptidase (leucine aminopeptidase), or carboxypeptidase
(carboxypeptidase A and B, procollagen C-proteinase). Chromogens
commonly used are 2-naphthylamine, 4-nitroaniline, and furylacrylic
acid. Assays are performed at room temperature (.about.25.degree.
C.) and contain an aliquot of the enzyme and the appropriate
substrate in a buffer. Reactions are carried out in an optical
cuvette and followed by the increase/decrease in absorbance of the
chromogen released during hydrolysis of the peptide substrate. The
change in absorbance is proportional to the enzyme activity in the
assay.
[0221] Enhancement of procollagen C-proteinase activity (HPRM-2) is
determined by measuring procollagen C-proteinase activity in the
absence and presence of enhancer protein. Procollagen C-proteinase
activity is measured as described above using an appropriate
carboxypeptidase substrate in the absence and in the presence of
varying amounts of HPRM-2. The increase in activity of procollagen
C-proteinase measured in the presence of HPRM-2 compared to that
measured in its absence is proportional to the activity of HPRM-2
in the assay.
[0222] XI Production of HPRM Specific Antibodies
[0223] HPRM is purified using PAGE electrophoresis
(Harrington(1990) Methods Enzymol 182:488-495), or other
purification techniques, is used to immunize rabbits and to produce
antibodies using standard protocols.
[0224] Alternatively, the HPRM amino acid sequence is analyzed
using LASERGENE software (DNASTAR) to determine regions of high
immunogenicity, and a corresponding oligopeptide is synthesized and
used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions are well described in
the art (Ausubel, supra, ch. 11).
[0225] Typically, oligopeptides 15 residues in length are
synthesized using a Model 431A peptide synthesizer (ABI) using
fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St.Louis Mo.) by
reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase immunogenicity (Ausubel, supra.). Rabbits are immunized
with the oligopeptide-KLH complex in complete Freund's adjuvant.
Resulting antisera are tested for antipeptide activity, for
example, by binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0226] XII Purification of Naturally Occurring HPRM Using Specific
Antibodies
[0227] Naturally occurring or recombinant HPRM is purified by
immunoaffinity chromatography using antibodies specific for HPRM.
An immunoaffinity column is constructed by covalently coupling
anti-HPRM antibody to an activated chromatographic resin, such as
CNBr-activated SEPHAROSE resin (APB). After the coupling, the resin
is blocked and washed according to the manufacturer's
instructions.
[0228] Media containing HPRM are passed over the immunoaffinity
column, and the column is washed under conditions, e.g., in high
ionic strength buffers in the presence of detergent, that allow the
preferential absorbance of HPRM. The column is eluted under
conditions, e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such as urea or thiocyanate ion, that disrupt
antibody/HPRM binding, and HPRM is collected.
[0229] XIII Identification of Molecules Which Interact with
HPRM
[0230] HPRM, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent (Bolton et al. (1973) Biochem
J 133:529-533). Candidate molecules previously arrayed in the wells
of a multi-well plate are incubated with the labeled HPRM, washed,
and any wells with labeled HPRM complex are assayed. Data obtained
using different concentrations of HPRM are used to calculate values
for the number, affinity, and association of HPRM with the
candidate molecules.
[0231] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
9 1 248 PRT Homo sapiens misc_feature Incyte ID No 456855 1 Met Phe
Leu Ala Lys Ala Leu Leu Glu Gly Ala Asp Arg Gly Leu 1 5 10 15 Gly
Glu Ala Leu Gly Gly Leu Phe Gly Gly Gly Gly Gln Arg Arg 20 25 30
Glu Gly Gly Gly Arg Asn Ile Gly Gly Ile Val Gly Gly Ile Val 35 40
45 Asn Phe Ile Ser Glu Ala Ala Ala Ala Gln Tyr Thr Pro Glu Pro 50
55 60 Pro Pro Thr Gln Gln His Phe Thr Ser Val Glu Ala Ser Glu Ser
65 70 75 Glu Glu Val Arg Arg Phe Arg Gln Gln Phe Thr Gln Leu Ala
Gly 80 85 90 Pro Asp Met Glu Val Gly Ala Thr Asp Leu Met Asn Ile
Leu Asn 95 100 105 Lys Val Leu Ser Lys His Lys Asp Leu Lys Thr Asp
Gly Phe Ser 110 115 120 Leu Asp Thr Cys Arg Ser Ile Val Ser Val Met
Asp Ser Asp Thr 125 130 135 Thr Gly Lys Leu Gly Phe Glu Glu Phe Lys
Tyr Leu Trp Asn Asn 140 145 150 Ile Lys Lys Trp Gln Cys Val Tyr Lys
Gln Tyr Asp Arg Asp His 155 160 165 Ser Gly Ser Leu Gly Ser Ser Gln
Leu Arg Gly Ala Leu Gln Ala 170 175 180 Ala Gly Phe Gln Leu Asn Glu
Gln Leu Tyr Gln Met Ile Val Arg 185 190 195 Arg Tyr Ala Asn Glu Asp
Gly Asp Met Asp Phe Asn Asn Phe Ile 200 205 210 Ser Cys Leu Val Arg
Leu Asp Ala Met Phe Arg Ala Phe Lys Ser 215 220 225 Leu Asp Arg Asp
Arg Asp Gly Leu Ile Gln Val Ser Ile Lys Glu 230 235 240 Trp Leu Gln
Leu Thr Met Tyr Ser 245 2 415 PRT Homo sapiens misc_feature Incyte
ID No 947429 2 Met Arg Gly Ala Asn Ala Trp Ala Pro Leu Cys Leu Leu
Leu Ala 1 5 10 15 Ala Ala Thr Gln Leu Ser Arg Gln Gln Ser Pro Glu
Arg Pro Val 20 25 30 Phe Thr Cys Gly Gly Ile Leu Thr Gly Glu Ser
Gly Phe Ile Gly 35 40 45 Ser Glu Gly Phe Pro Gly Val Tyr Pro Pro
Asn Ser Lys Cys Thr 50 55 60 Trp Lys Ile Thr Val Pro Glu Gly Lys
Val Val Val Leu Asn Phe 65 70 75 Arg Phe Ile Asp Leu Glu Ser Asp
Asn Leu Cys Arg Tyr Asp Phe 80 85 90 Val Asp Val Tyr Asn Gly His
Ala Asn Gly Gln Arg Ile Gly Arg 95 100 105 Phe Cys Gly Thr Phe Arg
Pro Gly Ala Leu Val Ser Ser Gly Asn 110 115 120 Lys Met Met Val Gln
Met Ile Phe Asp Ala Asn Thr Ala Gly Asn 125 130 135 Gly Phe Met Ala
Met Phe Ser Ala Ala Glu Pro Asn Glu Arg Gly 140 145 150 Asp Gln Tyr
Cys Gly Gly Leu Leu Asp Arg Pro Ser Gly Ser Phe 155 160 165 Lys Thr
Pro Asn Trp Pro Asp Arg Asp Tyr Pro Ala Gly Val Thr 170 175 180 Cys
Val Trp His Ile Val Ala Pro Lys Asn Gln Leu Ile Glu Leu 185 190 195
Lys Phe Glu Lys Phe Asp Val Glu Arg Asp Asn Tyr Cys Arg Tyr 200 205
210 Asp Tyr Val Ala Val Phe Asn Gly Gly Glu Val Asn Asp Ala Arg 215
220 225 Arg Ile Gly Lys Tyr Cys Gly Asp Ser Pro Pro Ala Pro Ile Val
230 235 240 Ser Glu Arg Asn Glu Leu Leu Ile Gln Phe Leu Ser Asp Leu
Ser 245 250 255 Leu Thr Ala Asp Gly Phe Ile Gly His Tyr Ile Phe Arg
Pro Lys 260 265 270 Lys Leu Pro Thr Thr Thr Glu Gln Pro Val Thr Thr
Thr Phe Pro 275 280 285 Val Thr Thr Gly Leu Lys Pro Thr Val Ala Leu
Cys Gln Gln Lys 290 295 300 Cys Arg Arg Thr Gly Thr Leu Glu Gly Asn
Tyr Cys Ser Ser Asp 305 310 315 Phe Val Leu Ala Gly Thr Val Ile Thr
Thr Ile Thr Arg Asp Gly 320 325 330 Ser Leu His Ala Thr Val Ser Ile
Ile Asn Ile Tyr Lys Glu Gly 335 340 345 Asn Leu Ala Ile Gln Gln Ala
Gly Lys Asn Met Ser Ala Arg Leu 350 355 360 Thr Val Val Cys Lys Gln
Cys Pro Leu Leu Arg Arg Gly Leu Asn 365 370 375 Tyr Ile Ile Met Gly
Gln Val Gly Glu Asp Gly Arg Gly Lys Ile 380 385 390 Met Pro Asn Ser
Phe Ile Met Met Phe Lys Thr Lys Asn Gln Lys 395 400 405 Leu Leu Asp
Ala Leu Lys Asn Lys Gln Cys 410 415 3 349 PRT Homo sapiens
misc_feature Incyte ID No 1515165 3 Met Lys Thr Leu Leu Leu Leu Leu
Leu Val Leu Leu Glu Leu Gly 1 5 10 15 Glu Ala Gln Gly Ser Leu His
Arg Val Pro Leu Arg Arg His Pro 20 25 30 Ser Leu Lys Lys Lys Leu
Arg Ala Arg Ser Gln Leu Ser Glu Phe 35 40 45 Trp Lys Ser His Asn
Leu Asp Met Ile Gln Phe Thr Glu Ser Cys 50 55 60 Ser Met Asp Gln
Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu Asp 65 70 75 Met Glu Tyr
Phe Gly Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn 80 85 90 Phe Thr
Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro 95 100 105 Ser
Val Tyr Cys Thr Ser Pro Ala Cys Lys Thr His Ser Arg Phe 110 115 120
Gln Pro Ser Gln Ser Ser Thr Tyr Ser Gln Pro Gly Gln Ser Phe 125 130
135 Ser Ile Gln Tyr Gly Thr Gly Ser Leu Ser Gly Ile Ile Gly Ala 140
145 150 Asp Gln Val Ser Val Glu Gly Leu Thr Val Val Gly Gln Gln Phe
155 160 165 Gly Glu Ser Val Thr Glu Pro Gly Gln Thr Phe Val Asp Ala
Glu 170 175 180 Phe Asp Gly Ile Leu Gly Leu Gly Tyr Pro Ser Leu Ala
Val Gly 185 190 195 Gly Val Thr Pro Val Phe Asp Asn Met Met Ala Gln
Asn Leu Val 200 205 210 Asp Leu Pro Met Phe Ser Val Tyr Met Ser Ser
Asn Pro Glu Gly 215 220 225 Gly Ala Gly Ser Glu Leu Ile Phe Gly Gly
Tyr Asp His Ser His 230 235 240 Phe Ser Gly Ser Leu Asn Trp Val Pro
Val Thr Lys Gln Ala Tyr 245 250 255 Trp Gln Ile Ala Leu Asp Asn Tyr
Ala Val Glu Cys Ala Asn Leu 260 265 270 Asn Val Met Pro Asp Val Thr
Phe Thr Ile Asn Gly Val Pro Tyr 275 280 285 Thr Leu Ser Pro Thr Ala
Tyr Thr Leu Leu Asp Phe Val Asp Gly 290 295 300 Met Gln Phe Cys Ser
Ser Gly Phe Gln Gly Leu Asp Ile His Pro 305 310 315 Pro Ala Gly Pro
Leu Trp Ile Leu Gly Asp Val Phe Ile Arg Gln 320 325 330 Phe Tyr Ser
Val Phe Asp Arg Gly Asn Asn Arg Val Gly Leu Ala 335 340 345 Pro Ala
Val Pro 4 1000 DNA Homo sapiens misc_feature Incyte ID No 456855 4
ttttttcata ccatctctaa gattgctgcc gcatttgctt gttaaactga aagcatgttt
60 cttgcaaagg ctctattgga aggagcagat cgaggtcttg gagaagctct
tggaggcctc 120 tttggaggag gtggtcagag aagagaagga ggaggaagaa
atattggagg gatagttgga 180 ggaattgtga attttatcag tgaggctgca
gcagctcagt atactccaga accgcctccc 240 actcagcagc atttcaccag
tgtggaggcc tcagaaagtg aggaagttag gcgatttcgg 300 caacaattta
cacagctggc tggaccagac atggaggtgg gtgccactga tctgatgaat 360
attctcaaca aagtcctttc taagcacaaa gatcttaaga ctgacggttt tagtcttgac
420 acctgccgga gcattgtgtc tgtcatggac agtgacacga ctggtaagct
gggctttgaa 480 gaatttaagt atctgtggaa caacatcaag aaatggcagt
gtgtttataa gcagtatgac 540 agggaccatt ctgggtctct gggaagttct
cagctgcggg gagctctgca ggccgcaggc 600 ttccagctaa atgaacaact
ttaccaaatg attgtccgcc ggtatgctaa tgaagatgga 660 gatatggatt
ttaacaattt catcagctgc ttggtccgcc tggatgccat gtttcgtgcc 720
ttcaagtctc tggatagaga tagagatggc ctgattcaag tgtctatcaa agagtggctg
780 cagttgacca tgtattcctg aagtgggaac tgagaagtca agatcctccc
tggaggacag 840 gactgaaaac cttgccaagc tgtacacagt tgctgatacc
ctgtgcaaca gctctcattt 900 cctggcaagc tctttcacaa ccctacatat
ttctgatcat gtgctgcctt ttactgctga 960 attaaaacag atatttcacg
aaaaaaaaaa aaaaaaaaaa 1000 5 1802 DNA Homo sapiens misc_feature
Incyte ID No 947429 5 cgctgtcggt gcggcggcgc gcgtgcgggt gcaaacccga
gcgtctacgc tgccatgagg 60 ggcgcgaacg cctgggcgcc actctgcctg
ctgctggctg ccgccaccca gctctcgcgg 120 cagcagtccc cagagagacc
tgttttcaca tgtggtggca ttcttactgg agagtctgga 180 tttattggca
gtgaaggttt tcctggagtg taccctccaa atagcaaatg tacttggaaa 240
atcacagttc ccgaaggaaa agtagtcgtt ctcaatttcc gattcataga cctcgagagt
300 gacaacctgt gccgctatga ctttgtggat gtgtacaatg gccatgccaa
tggccagcgc 360 attggccgct tctgtggcac tttccggcct ggagcccttg
tgtccagtgg caacaagatg 420 atggtgcaga tgatttttga tgccaacaca
gctggcaatg gcttcatggc catgttctcc 480 gctgctgaac caaacgaaag
aggggatcag tattgtggag gactccttga cagaccttcc 540 ggctctttta
aaacccccaa ctggccagac cgggattacc ctgcaggagt cacttgtgtg 600
tggcacattg tagccccaaa gaatcagctt atagaattaa agtttgagaa gtttgatgtg
660 gagcgagata actactgccg atatgattat gtggctgtgt ttaatggcgg
ggaagtcaac 720 gatgctagaa gaattggaaa gtattgtggt gatagtccac
ctgcgccaat tgtgtctgag 780 agaaatgaac ttcttattca gtttttatca
gacttaagtt taactgcaga tgggtttatt 840 ggtcactaca tattcaggcc
aaaaaaactg cctacaacta cagaacagcc tgtcaccacc 900 acattccctg
taaccacggg tttaaaaccc accgtggcct tgtgtcaaca aaagtgtaga 960
cggacgggga ctctggaggg caattattgt tcaagtgact ttgtattagc cggcactgtt
1020 atcacaacca tcactcgcga tgggagtttg cacgccacag tctcgatcat
caacatctac 1080 aaagagggaa atttggcgat tcagcaggcg ggcaagaaca
tgagtgccag gctgactgtc 1140 gtctgcaagc agtgccctct cctcagaaga
ggtctaaatt acattattat gggccaagta 1200 ggtgaagatg ggcgaggcaa
aatcatgcca aacagcttta tcatgatgtt caagaccaag 1260 aatcagaagc
tcctggatgc cttaaaaaat aagcaatgtt aacagtgaac tgtgtccatt 1320
taagctgtat tctgccattg cctttgaaag atctatgttc tctcagtaga aaaaaaaata
1380 cttataaaat tacatattct gaaagaggat tccgaaagat gggactggtt
gactcttcac 1440 atgatggagg tatgaggcct ccgagatagc tgagggaagt
tctttgcctg ctgtcagagg 1500 agcagctatc tgattggaaa cctgccgact
tagtgcggtg ataggaagct aaaagtgtca 1560 agcgttgaca gcttggaagc
gtttatttat acatctctgt aaaaggatat tttagaattg 1620 agttgtgtga
agatgtcaaa aaaagatttt agaagtgcaa tatttatagt gttatttgtt 1680
tcaccttcaa gcctttgccc tgaggtgtta caatcttgtc ttgcgttttc taaatcaatg
1740 cttaataaaa tatttttaaa ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaacga 1800 at 1802 6 2073 DNA Homo sapiens misc_feature Incyte
ID No 1515165 6 cggagggggc aagggagaag ctgctggtcg gactcacaat
gaaaacgctc cttcttttgc 60 tgctggtgct cctggagctg ggagaggccc
aaggatccct tcacagggtg cccctcagga 120 ggcatccgtc cctcaagaag
aagctgcggg cacggagcca gctctctgag ttctggaaat 180 cccataattt
ggacatgatc cagttcaccg agtcctgctc aatggaccag agtgccaagg 240
aacccctcat caactacttg gatatggaat acttcggcac tatctccatt ggctccccac
300 cacagaactt cactgtcatc ttcgacactg gctcctccaa cctctgggtc
ccctctgtgt 360 actgcactag cccagcctgc aagacgcaca gcaggttcca
gccttcccag tccagcacat 420 acagccagcc aggtcaatct ttctccattc
agtatggaac cgggagcttg tccgggatca 480 ttggagccga ccaagtctct
gtggaaggac taaccgtggt tggccagcag tttggagaaa 540 gtgtcacaga
gccaggccag acctttgtgg atgcagagtt tgatggaatt ctgggcctgg 600
gatacccctc cttggctgtg ggaggagtga ctccagtatt tgacaacatg atggctcaga
660 acctggtgga cttgccgatg ttttctgtct acatgagcag taacccagaa
ggtggtgccg 720 ggagcgagct gatttttgga ggctacgacc actcccattt
ctctgggagc ctgaattggg 780 tcccagtcac caagcaagct tactggcaga
ttgcactgga taactatgct gtggagtgtg 840 ccaaccttaa cgtcatgccg
gatgtcacct tcaccattaa cggagtcccc tataccctca 900 gcccaactgc
ctacacccta ctggacttcg tggatggaat gcagttctgc agcagtggct 960
ttcaaggact tgacatccac cctccagctg ggcccctctg gatcctgggg gatgtcttca
1020 ttcgacagtt ttactcagtc tttgaccgtg ggaataaccg tgtgggactg
gccccagcag 1080 tcccctaagg aggggccttg tgtctgtgcc tgcctgtctg
acagaccttg aatatgttag 1140 gctggggcat tctttacacc tacaaaaagt
tattttccag agaatgtagc tgtttccagg 1200 gttgcaactt gaattaagac
caaacagaac atgagaatac acacacacac acacatatac 1260 acacacacac
acttcacaca tacacaccac tcccaccacc gtcatgatgg aggaattacg 1320
ttatacattc atattttgta ttgatttttg attatgaaaa tcaaaaattt tcacatttga
1380 ttatgaaaat ctccaaacat atgcacaagc agagatcatg gtataataaa
tccctttgca 1440 actccactca gccctgacaa cccatccaca cacggccagg
cctgtttatc tacactgctg 1500 cccactcctc tctccagctc cacatgctgt
acctggatca ttctgaagca aattccgagc 1560 attacatcat tttgtccata
aatatttcta acatccttaa atatacaatc ggaattcaag 1620 catctcccat
tgtcccacaa atgtttggct gtttttgtag ttggattgtt tgtattagga 1680
ttcaagcaag gcccatatat tgcatttatt tgaaatgtct gtaagtctct ttccatctac
1740 agagtttagc acatttgaac gttgctggtt gaaatcccga ggtgtcattt
gacatggttc 1800 tctgaactta tctttcctat aaaatggtag ttagatctgg
aggtctgatt ttgtggcaaa 1860 aatacttcct aggtggtgct gggtacttct
tgttgcatcc tgtcaggagg cagataatgc 1920 tggtgcctct ctattggtaa
tgttaagact gctgggtggg tttggagttc ttggctttaa 1980 tcattcatta
caaagttcag cattttaaaa aaaaaaaaaa aaaaggaaaa aagaaagaaa 2040
aagagaaaaa agaaaaaaaa aaggaaagag ggg 2073 7 266 PRT Homo sapiens
misc_feature Incyte ID No 164403 7 Met Phe Leu Val Asn Ser Phe Leu
Lys Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Leu Gly
Gly Gly Leu Gly Asn Val Leu Gly 20 25 30 Gly Leu Ile Ser Gly Ala
Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Gly Gly Gly
Gly Gly Gly Gly Thr Ala Met Arg Ile Leu 50 55 60 Gly Gly Val Ile
Ser Ala Ile Ser Glu Ala Ala Ala Gln Tyr Asn 65 70 75 Pro Glu Pro
Pro Pro Pro Arg Thr His Tyr Ser Asn Ile Glu Ala 80 85 90 Asn Glu
Ser Glu Glu Val Arg Gln Phe Arg Arg Leu Phe Ala Gln 95 100 105 Leu
Ala Gly Asp Asp Met Glu Val Ser Ala Thr Glu Leu Met Asn 110 115 120
Ile Leu Asn Lys Val Val Thr Arg His Pro Asp Leu Lys Thr Asp 125 130
135 Gly Phe Gly Ile Asp Thr Cys Arg Ser Met Val Ala Val Met Asp 140
145 150 Ser Asp Thr Thr Gly Lys Leu Gly Phe Glu Glu Phe Lys Tyr Leu
155 160 165 Trp Asn Asn Ile Lys Lys Trp Gln Ala Ile Tyr Lys Gln Phe
Asp 170 175 180 Val Asp Arg Ser Gly Thr Ile Gly Ser Ser Glu Leu Pro
Gly Ala 185 190 195 Phe Glu Ala Ala Gly Phe His Leu Asn Glu His Leu
Tyr Ser Met 200 205 210 Ile Ile Arg Arg Tyr Ser Asp Glu Gly Gly Asn
Met Asp Phe Asp 215 220 225 Asn Phe Ile Ser Cys Leu Val Arg Leu Asp
Ala Met Phe Arg Ala 230 235 240 Phe Lys Ser Leu Asp Lys Asp Gly Thr
Gly Gln Ile Gln Val Asn 245 250 255 Ile Gln Glu Trp Leu Gln Leu Thr
Met Tyr Ser 260 265 8 468 PRT Homo sapiens misc_feature Incyte ID
No 2589009 8 Met Leu Pro Ala Ala Leu Thr Ser Phe Leu Gly Pro Phe
Leu Leu 1 5 10 15 Ala Trp Val Leu Pro Leu Ala Arg Gly Gln Thr Pro
Asn Tyr Thr 20 25 30 Arg Pro Val Phe Leu Cys Gly Gly Asp Val Thr
Gly Glu Ser Gly 35 40 45 Tyr Val Ala Ser Glu Gly Phe Pro Asn Leu
Tyr Pro Pro Asn Lys 50 55 60 Lys Cys Ile Trp Thr Ile Thr Val Pro
Glu Gly Gln Thr Val Ser 65 70 75 Leu Ser Phe Arg Val Phe Asp Met
Glu Leu His Pro Ser Cys Arg 80 85 90 Tyr Asp Ala Leu Glu Val Phe
Ala Gly Ser Gly Thr Ser Gly Gln 95 100 105 Arg Leu Gly Arg Phe Cys
Gly Thr Phe Arg Pro Ala Pro Val Val 110 115 120 Ala Pro Gly Asn Gln
Val Thr Leu Arg Met Thr Thr Asp Glu Gly 125 130 135 Thr Gly Gly Arg
Gly Phe Leu Leu Trp Tyr Ser Gly Arg Ala Thr 140 145 150 Ser Gly Thr
Glu His Gln Phe Cys Gly Gly Arg Met Glu Lys Ala 155 160 165 Gln Gly
Thr Leu Thr Thr Pro Asn Trp Pro Glu Ser Asp Tyr Pro 170 175 180 Pro
Gly Ile Ser Cys Ser Trp His Ile Ile Ala Pro Ser Asn Gln 185 190 195
Val Ile Met Leu Thr Phe Gly
Lys Phe Asp Val Glu Pro Asp Thr 200 205 210 Tyr Cys Arg Tyr Asp Ser
Val Ser Val Phe Asn Gly Ala Val Ser 215 220 225 Asp Asp Ser Lys Arg
Leu Gly Lys Phe Cys Gly Asp Lys Ala Pro 230 235 240 Ser Pro Ile Ser
Ser Glu Gly Asn Glu Leu Leu Val Gln Phe Val 245 250 255 Ser Asp Leu
Ser Val Thr Ala Asp Gly Phe Ser Ala Ser Tyr Arg 260 265 270 Thr Leu
Pro Arg Asp Ala Val Glu Lys Glu Ser Ala Leu Ser Pro 275 280 285 Gly
Glu Asp Val Gln Arg Gly Pro Gln Ser Arg Ser Asp Pro Lys 290 295 300
Thr Gly Thr Gly Pro Lys Val Lys Pro Pro Thr Lys Pro Lys Ser 305 310
315 Gln Pro Ala Glu Thr Pro Glu Ala Ser Pro Ala Thr Gln Ala Thr 320
325 330 Pro Val Ala Pro Ala Ala Pro Ser Ile Thr Cys Pro Lys Gln Tyr
335 340 345 Lys Arg Ser Gly Thr Leu Gln Ser Asn Phe Cys Ser Ser Ser
Leu 350 355 360 Val Val Thr Gly Thr Val Lys Thr Met Val Arg Gly Pro
Gly Glu 365 370 375 Gly Leu Thr Val Thr Val Ser Leu Leu Gly Val Tyr
Lys Thr Gly 380 385 390 Gly Leu Asp Leu Pro Ser Pro Pro Ser Gly Thr
Ser Leu Lys Leu 395 400 405 Tyr Val Pro Cys Arg Gln Met Pro Pro Met
Lys Lys Gly Ala Ser 410 415 420 Tyr Leu Leu Met Gly Gln Val Glu Glu
Asn Arg Gly Pro Ile Leu 425 430 435 Pro Pro Glu Ser Phe Val Val Leu
Tyr Arg Ser Asn Gln Asp Gln 440 445 450 Ile Leu Asn Asn Leu Ser Lys
Arg Lys Cys Pro Ser Gln Pro Arg 455 460 465 Thr Ala Ala 9 396 PRT
Homo sapiens misc_feature Incyte ID No 181994 9 Met Lys Thr Leu Leu
Leu Leu Leu Leu Val Leu Leu Glu Leu Gly 1 5 10 15 Glu Ala Gln Gly
Ser Leu His Arg Val Pro Leu Arg Arg His Pro 20 25 30 Ser Leu Lys
Lys Lys Leu Arg Ala Arg Ser Gln Leu Ser Glu Phe 35 40 45 Trp Lys
Ser His Asn Leu Asp Met Ile Gln Phe Thr Glu Ser Cys 50 55 60 Ser
Met Asp Gln Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu Asp 65 70 75
Met Glu Tyr Phe Gly Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn 80 85
90 Phe Thr Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro 95
100 105 Ser Val Tyr Cys Thr Ser Pro Ala Cys Lys Thr His Ser Arg Phe
110 115 120 Gln Pro Ser Gln Ser Ser Thr Tyr Ser Gln Pro Gly Gln Ser
Phe 125 130 135 Ser Ile Gln Tyr Gly Thr Gly Ser Leu Ser Gly Ile Ile
Gly Ala 140 145 150 Asp Gln Val Ser Val Glu Gly Leu Thr Val Val Gly
Gln Gln Phe 155 160 165 Gly Glu Ser Val Thr Glu Pro Gly Gln Thr Phe
Val Asp Ala Glu 170 175 180 Phe Asp Gly Ile Leu Gly Leu Gly Tyr Pro
Ser Leu Ala Val Gly 185 190 195 Gly Val Thr Pro Val Phe Asp Asn Met
Met Ala Gln Asn Leu Val 200 205 210 Asp Leu Pro Met Phe Ser Val Tyr
Met Ser Ser Asn Pro Glu Gly 215 220 225 Gly Ala Gly Ser Glu Leu Ile
Phe Gly Gly Tyr Asp His Ser His 230 235 240 Phe Ser Gly Ser Leu Asn
Trp Val Pro Val Thr Lys Gln Ala Tyr 245 250 255 Trp Gln Ile Ala Leu
Asp Asn Ile Gln Val Gly Gly Thr Val Met 260 265 270 Phe Cys Ser Glu
Gly Cys Gln Ala Ile Val Asp Thr Gly Thr Ser 275 280 285 Leu Ile Thr
Gly Pro Ser Asp Lys Ile Lys Gln Leu Gln Asn Ala 290 295 300 Ile Gly
Ala Ala Pro Val Asp Gly Glu Tyr Ala Val Glu Cys Ala 305 310 315 Asn
Leu Asn Val Met Pro Asp Val Thr Phe Thr Ile Asn Gly Val 320 325 330
Pro Tyr Thr Leu Ser Pro Thr Ala Tyr Thr Leu Leu Asp Phe Val 335 340
345 Asp Gly Met Gln Phe Cys Ser Ser Gly Phe Gln Gly Leu Asp Ile 350
355 360 His Pro Pro Ala Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Ile
365 370 375 Arg Gln Phe Tyr Ser Val Phe Asp Arg Gly Asn Asn Arg Val
Gly 380 385 390 Leu Ala Pro Ala Val Pro 395
* * * * *