U.S. patent application number 10/652893 was filed with the patent office on 2005-11-10 for mammalian proteases; related reagents.
This patent application is currently assigned to Schering Corporation, a New Jersey corporation. Invention is credited to Balasubramanian, Sriram, Ford, John, Gorman, Daniel M., Zurawski, Gerard.
Application Number | 20050249733 10/652893 |
Document ID | / |
Family ID | 24836687 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249733 |
Kind Code |
A1 |
Balasubramanian, Sriram ; et
al. |
November 10, 2005 |
Mammalian proteases; related reagents
Abstract
Nucleic acids encoding various proteases, from a mammal,
reagents related thereto, including specific antibodies, and
purified proteins are described. Methods of using said reagents and
related diagnostic kits are also provided.
Inventors: |
Balasubramanian, Sriram; (La
Jolla, CA) ; Ford, John; (Palo Alto, CA) ;
Gorman, Daniel M.; (Palo Alto, CA) ; Zurawski,
Gerard; (Midlothian, CA) |
Correspondence
Address: |
DNAX RESEARCH, INC.
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation, a New Jersey
corporation
|
Family ID: |
24836687 |
Appl. No.: |
10/652893 |
Filed: |
August 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652893 |
Aug 29, 2003 |
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09650284 |
Aug 29, 2000 |
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6638507 |
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09650284 |
Aug 29, 2000 |
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08706216 |
Aug 30, 1996 |
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6140098 |
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Current U.S.
Class: |
424/145.1 ;
435/7.2 |
Current CPC
Class: |
Y10S 435/81 20130101;
C07K 16/40 20130101; C12N 9/6421 20130101; C12N 9/64 20130101; G01N
2333/96425 20130101 |
Class at
Publication: |
424/145.1 ;
435/007.2 |
International
Class: |
A61K 039/395; G01N
033/53; G01N 033/567 |
Claims
1-20. (canceled)
21. A substantially pure polypeptide comprising the amino acid
sequence of SEQ ID NO: 6, or a fragment thereof.
22. The polypeptide of claim 21, wherein the fragment is at least
30 amino acids.
23. The polypeptide of claim 21, where in the polypeptide comprises
residues 20 to 391 of SEQ ID NO: 6.
24. The polypeptide of claim 21 fused to a second heterologous
polypeptide.
25. The polypeptide of claim 24, wherein the second heterologous
polypeptide is a reporter polypeptide.
26. The polypeptide of claim 25, wherein the reporter polypeptide
is selected from the group consisting of luceferase,
.beta.-galactosidase, trpE, Protein A, .beta.-lactamase,
.alpha.-amlyase, alcohol dehydrogenase, and yeast .alpha. mating
factor.
27. The polypeptide of claim 21, wherein the polypeptide is a
soluble polypeptide.
28. The polypeptide of claim 21, wherein the polypeptide is
recombinantly produced.
29. The polypeptide of claim 21, wherein the polypeptide is a
naturally occurring polypeptide.
30. The polypeptide of claim 21, wherein the fragment comprises a
proteolytically active portion of SEQ ID NO: 6.
Description
FIELD OF THE INVENTION
[0001] The present invention contemplates compositions related to
proteins from animals, e.g., mammals, which function as proteases.
In particular, it provides nucleic acids which encode, antibodies
to, and proteins which exhibit biological functions, e.g., capacity
to degrade proteinaceous substrates.
BACKGROUND OF THE INVENTION
[0002] The proteases are a very broad group of enzymes which carry
out an enzymatic function of hydrolysing a peptide bond. Within the
group, there is a wide range of substrate specificities for the
amino acids adjacent the cleavage sites. Proteases are typically
categorized on the basis of their catalytic mecahnisms, e.g., based
upon studies of their active sites, or by the effects of pH. Four
main categories of proteases are serine proteinases, sulfhydryl
proteases, acid proteases, and metalloproteases. They may also be
classified according to their cleavage sites, e.g., endoproteases,
amino peptidases, or carboxy peptidases.
[0003] Proteases have traditionally held a large share of the
industrial enzyme market. Proteases are used in many industrial
processes, including in detergents and cleaning products, e.g., to
degrade protein materials such as blood and stains, in leather
production, e.g., to remove hair, in baking, e.g., to break down
glutens, in flavorings, e.g., soy sauce, in meat tenderizing, e.g.,
to break down collagen, in gelatin or food supplement production,
in the textile industry, in waste treatment, and in the
photographic industry. See, e.g., Gusek (1991) Inform 1:14-18;
Zamost, et al. (1996) J. Industrial Microbiol, 8:71-82; James and
Simpson (1996) CRC Critical Reviews in Food Science and Nutrition
36:437-463; Teichgraeber, et al. (1993) Trends in Food Science and
Technology 4:145-149; Tjwan, et al. (1993) J. Dairy Research
60:269-286; Haard (1992) J. Aguatic Food Product Technology
1:17-35; van Dijk (1995) Laundry and Cleaning News 21:32-33; Nolte,
et al. (1996) J. Textile Institute 87:212-226; Chikkodi, et al.
(1995) Textile Res. J. 65:564-569; and Shih (1993) Poultry Science
72:1617-1620.
[0004] While there are many uses for proteases, there is always the
need for a more active protease under various specific conditions.
There is a need for new proteinases of differing properties,
specificities, and activities.
SUMMARY OF THE INVENTION
[0005] The present invention provides a composition of matter
selected from an antibody binding site which specifically binds to
a human APG04, FDH02, or D1B2 protein or significant fragment
thereof; an expression vector encoding a human APG04, FDH02, or
D1B2 protein or significant fragment thereof; a substantially pure
protein which is specifically recognized by the antibody binding
site; and a substantially pure APG04, FDH02, or D1B2 protein or
peptide thereof, or a fusion protein comprising at least a 30 amino
acid fragment of human APG04, FDH02, or D1B2 protein sequence.
[0006] In the antibody binding site embodiments, the antibody
binding site may be: specifically immunoreactive with a mature
protein selected from the group consisting of the polypeptides of
SEQ ID NO: 2, 4, and 6; raised against a purified or recombinantly
produced human APG04, FDH02, or D1B2 protein; in a monoclonal
antibody, Fab, or F(ab)2; or in a detectably labeled antibody. In
certain embodiments; the antibody binding site is detected in a
biological sample by a method of: contacting a binding agent having
an affinity for the human APG04, FDH02, or D1B2 protein with the
biological sample; incubating the binding agent with the biological
sample to form a binding agent:human APG04, FDH02, or D1B2 protein
complex; and detecting the complex. In a preferred embodiment, the
biological sample is from a human, and the binding agent is an
antibody.
[0007] A kit embodiment is provided comprising a composition,
described above, with either instructional material for the use
and/or disposal of the composition and products; or segregation of
the composition into a compartment or container.
[0008] A nucleic acid embodiment of the invention includes an
expression vector encoding a human APG04, FDH02, or D1B2 protein,
wherein the protein specifically binds an antibody generated
against an immunogen selected from the mature polypeptide portions
of SEQ ID NO: 2, 4, and 6. The vector may: encode a human APG04,
FDH02, or D1B2 polypeptide with complete sequence identity to a
naturally occurring human APG04, FDH02, or D1B2 protein; encode a
human APG04, FDH02, or D1B2 protein comprising sequence selected
from the polypeptides of SEQ ID NO: 2, 4, and 6; or comprise
sequence selected from the nucleic acids of SEQ ID NO: 1, 3, or 5.
In other embodiments, the vector is capable of selectively
hybridizing to a nucleic acid encoding a natural human APG04,
FDH02, or D1B2 protein, e.g., a mature protein coding segment of
SEQ ID NO: 1, 3, or 5. In various preferred embodiments, the
isolated nucleic acid is detected in a biological sample by a
method: contacting a biological sample with a nucleic acid probe
capable of selectively hybridizing to the nucleic acid; incubating
the nucleic acid probe with the biological sample to form a hybrid
of the nucleic acid probe with complementary nucleic acid sequences
present in the biological sample; and determining the extent of
hybridization of the nucleic acid probe to the complementary
nucleic acid sequences. In such method, preferably the nucleic acid
probe is capable of hybridizing to a nucleic acid encoding a
protein consisting of the polypeptides of SEQ ID NO: 2, 4, or
6.
[0009] In protein embodiments, the human APG04, FDH02, or D1B2
protein specifically binds to an antibody generated against the
respective immunogen; e.g., the polypeptides of SEQ ID NO: 2, 4, or
6. In various embodiments, the isolated human APG04, FDH02, or D1B2
protein consists of a polypeptide comprising sequence from SEQ ID
NO: 2, 4, or 6; is recombinantly produced, or is a naturally
occurring protein.
[0010] The present invention also embraces a cell transfected with
the isolated or recombinant nucleic acid encoding a human APG04,
FDH02, or D1B2 protein, e.g., where the nucleic acid has SEQ ID NO:
1, 3, or 5.
DETAILED DESCRIPTION
[0011] Outline
1 I. General II. Definitions III. Nucleic Acids IV. Making APG04,
FDH02, or D1B2 Protein V. Antibodies a. antibody production b.
immunoassays VI. Purified APG04, FDH02, and D1B2 Protein VII.
Physical Variants VIII. Binding Agent: APG04, FDH02, and D1B2
Protein Complexes IX. Functional Variants X. Uses XI. Kits XII.
Substrate Identification
[0012] I. General
[0013] The present invention provides DNA sequences encoding
mammalian proteins which exhibit structural properties or motifs
characteristic of a protease. The proteases described herein are
designated APG04, FDH02 and D1B2. See Tables 1, 2, and 3.
[0014] The descriptions below are directed, for exemplary purposes,
to primate embodiments, e.g., human, but are likewise applicable to
related embodiments from other, e.g., natural, sources. These
sources should, where appropriate, include various vertebrates,
typically warm blooded animals, e.g., birds and mammals,
particularly domestic animals, and primates.
2TABLE 1 Human APG04 nucleotide and predicted amino acid sequence.
SEQ ID NO: 1 and 2. The predicted leader sequence is underlined.
The mature peptide probably begins about at Pro (position 21).
Active site/zinc chelating residues are indicated by *. TTGATGGCCA
CCAGGTGATC TCTGGTCTCT TCAGTGTGGC 60 TTTGCAGACT ATAAAGGCGC
AGCGCGCCAA CGAGGCGGGT TGGCCCCAGA CGGCGGAGAG 120 GAAGGGCAGA
GTCGGCGGTC CTGAGACTTG GGGCGGCCCC TTGGAGGTCA GCCCCGCTCG 180
CTCCTCCCGG CCCTCTCCTC CTCTCCGAGG TCCGAGGCGG GCAGCGGGCT GTGGGCGGGC
240 AGGAGGCTGC GGAGGGGCGG GGGGCAGGAA GGGGCGGGGG GCTCGGCGCA
CTCGGCAGGA 300 AGAGACCGAC CCGCCACCCG CCGTAGCCCG CGCGCCCCTG
GCACTCAATC CCCGCC ATG 354 TGG GGG CTC CTG CTC Met 1 Trp Gly Leu Leu
Leu 5 GCC CTG GCC GCC TTC GCG CCG GCC GTC GGC CCG 402 GCT CTG GGG
GCG CCC Ala Leu Ala Ala Phe Ala Pro Ala Val Gly Pro 10 15 Ala Leu
Gly Ala Pro 20 AGG AAC TCG GTG CTG GGC CTC GCG CAG CCC GGG 450 ACC
ACC AAG GTC CCA Arg Asn Ser Val Leu Gly Leu Ala Gln Pro Gly 25 30
Thr Thr Lys Val Pro 35 GGC TCG ACC CCG GCC CTG CAT AGC AGC CCG GCA
498 CAG CCG CCG GCG GAG Gly Ser Thr Pro Ala Leu His Ser Ser Pro Ala
40 45 Gln Pro Pro Ala Glu 50 ACA GCT AAC GGG ACC TCA GAA CAG CAT
GTC CGG 546 ATT CGA GTC ATC AAG Thr Ala Asn Gly Thr Ser Glu Gln His
Val Arg 55 60 65 Ile Arg Val Ile Lys 70 AAG AAA AAG GTC ATT ATG AAG
AAG CGG AAG AAG 594 CTA ACT CTA ACT CGC Lys Lys Lys Val Ile Met Lys
Lys Arg Lys Lys 75 80 Leu Thr Leu Thr Arg 85 CCC ACC CCA CTG GTG
ACT GCC GGG CCC CTT GTG 642 ACC CCC ACT CCA GCA Pro Thr Pro Leu Val
Thr Ala Gly Pro Leu Val 90 95 Thr Pro Thr Pro Ala 100 GGG ACC CTC
GAC CCC GCT GAG AAA CAA GAA ACA 690 GGC TGT CCT CCT TTG Gly Thr Leu
Asp Pro Ala Glu Lys Gln Glu Thr 105 110 Gly Cys Pro Pro Leu 115 GGT
CTG GAG TCC CTG CGA GTT TCA GAT AGC CGG 738 CTT GAG GCA TCC AGC Gly
Leu Glu Ser Leu Arg Val Ser Asp Ser Arg 120 125 Leu Glu Ala Ser Ser
130 AGC CAG TCC TTT GGT CTT GGA CCA CAC CGA GGA 786 CGG CTC AAC ATT
CAG Ser Gln Ser Phe Gly Leu Gly Pro His Arg Gly 135 140 145 Arg Leu
Asn Ile Gln 150 TCA GGC CTG GAG GAC GGC GAT CTA TAT GAT GGA 834 GCC
TGG TGT GCT GAG Ser Gly Leu Glu Asp Gly Asp Leu Tyr Asp Gly 155 160
Ala Trp Cys Ala Glu 165 GAG CAG GAC GCC GAT CCA TGG TTT CAG GTG GAC
882 GCT GGG CAC CCC ACC Glu Gln Asp Ala Asp Pro Trp Phe Gln Val Asp
170 175 Ala Gly His Pro Thr 180 CGC TTC TCG GGT GTT ATC ACA CAG GGC
AGG AAC 930 TCT GTC TGG AGG TAT Arg Phe Ser Gly Val Ile Thr Gln Gly
Arg Asn 185 190 Ser Val Trp Arg Tyr 195 GAC TGG GTC ACA TCA TAC AAG
GTC CAG TTC AGC 978 AAT GAC AGT CGG ACC Asp Trp Val Thr Ser Tyr Lys
Val Gln Phe Ser 200 205 Asn Asp Ser Arg Thr 210 TGG TGG GGA AGT AGG
AAC CAC AGC AGT GGG ATG 1026 GAC GCA GTA TTT CCT Trp Trp Gly Ser
Arg Asn His Ser Ser Gly Met 215 220 225 Asp Ala Val Phe Pro 230 GCC
AAT TCA GAC CCA GAA ACT CCA GTG CTG AAC 1074 CTC CTG CCG GAG CCC
Ala Asn Ser Asp Pro Glu Thr Pro Val Leu Asn 235 240 Leu Leu Pro Glu
Pro 245 CAG GTG GCC CGC TTC ATT CGC CTG CTG CCC CAG 1122 ACC TGG
CTC CAG GGA Gln Val Ala Arg Phe Ile Arg Leu Leu Pro Gln 250 255 Thr
Trp Leu Gln Cly 260 GGC GCG CCT TGC CTC CGG GCA GAG ATC CTG GCC
1170 TGC CCA GTC TCA GAC Gly Ala Pro Cys Leu Arg Ala Glu Ile Leu
Ala 265 270 Cys Pro Val Ser Asp 275 CCC AAT GAC CTA TTC CTT GAG GCC
CCT GCG TCG 1218 GGA TCC TCT GAC CCT Pro Asn Asp Leu Phe Leu Glu
Ala Pro Ala Ser 280 285 Gly Ser Ser Asp Pro 290 CTA GAC TTT CAG CAT
CAC AAT TAC AAG GCC ATG 1266 AGG AAG CTG ATG AAG Leu Asp Phe Gln
His His Asn Tyr Lys Ala Met 295 300 305 Arg Lys Leu Met Lys 310 CAG
GTA CAA GAG CAA TGC CCC AAC ATC ACC CGC 1314 ATC TAC AGC ATT GGG
Gln Val Gln Glu Gln Cys Pro Asn Ile Thr Arg 315 320 Ile Tyr Ser Ile
Gly 325 AAG AGC TAC CAG GGC CTG AAG CTG TAT GTG ATG 1362 GAA ATG
TCG GAC AAG Lys Ser Tyr Gln Gly Leu Lys Leu Tyr Val Met 330 335 Glu
Met Ser Asp Lys 340 CCT GGG GAG CAT GAG CTG GGG GAG CCT GAG GTG
1410 CGC TAC GTG GCT GGC Pro Gly Glu His Glu Leu Gly Glu Pro Glu
Val 345 350 Arg Tyr Val Ala Gly 355 ATG CAT GGG AAC GAG GCC CTG GGG
CGG GAG TTG 1458 CTT CTG CTC CTG ATG Met His Gly Asn Glu Ala Leu
Gly Arg Glu Leu * * 360 365 Leu Leu Leu Leu Met 370 CAG TTC CTG TGC
CAT GAG TTC CTG CGA GGG AAC 1506 CCA CAG GTG ACC CGG Gln Phe Leu
Cys His Glu Phe Leu Arg Gly Asn * 375 380 385 Pro Gln Val Thr Arg
390 CTG CTC TCT GAG ATG CGC ATT CAC CTG CTG CCC 1554 TCC ATG AAC
CCT GAT Leu Leu Ser Glu Met Arg Ile His Leu Leu Pro 395 400 Ser Met
Asn Pro Asp 405 GGC TAT GAG ATC GCC TAC CAC CGG GGT TCA GAG 1602
CTG GTG GGC TGG GCC Gly Tyr Glu Ile Ala Tyr His Arg Gly Ser Glu 410
415 Leu Val Gly Trp Ala 420 GAG GGC CGC TGG AAC AAC CAG AGC ATC GAT
CTT 1650 AAC CAT AAT TTT GCT Glu Gly Arg Trp Asn Asn Gln Ser Ile
Asp Leu 425 430 Asn His Asn Phe Ala 435 GAC CTC AAC ACA CCA CTG TGG
GAA GCA CAG GAC 1698 GAT GGG AAG GTG CCC Asp Leu Asn Thr Pro Leu
Trp Glu Ala Gln Asp 440 445 Asp Gly Lys Val Pro 450 CAC ATC GTC CCC
AAC CAT CAC CTG CCA TTG CCC 1746 ACT TAC TAC ACC CTG His Ile Val
Pro Asn His His Leu Pro Leu Pro 455 460 465 Thr Tyr Tyr Thr Leu 470
CCC AAT GCC ACC GTG GCT CCT GAA ACG CGG GCA 1794 GTA ATC AAG TGG
ATG Pro Asn Ala Thr Val Ala Pro Glu Thr Arg Ala 475 480 Val Ile Lys
Trp Met 485 AAG CGG ATC CCC TTT GTG CTA AGT GCC AAC CTC 1842 CAC
GGG GGT GAG CTC Lys Arg Ile Pro Phe Val Leu Ser Ala Asn Leu 490 495
His Gly Gly Glu Leu * 500 GTG GTG TCC TAC CCA TTC GAC ATG ACT CGC
ACC 1890 CCG TGG GCT GCC CGC Val Val Ser Tyr Pro Phe Asp Met Thr
Arg Thr 505 510 Pro Trp Ala Ala Arg 515 GAG CTC ACG CCC ACA CCA GAT
GAT GCT GTG TTT 1938 CGC TGG CTC AGC ACT Glu Leu Thr Pro Thr Pro
Asp Asp Ala Val Phe 520 525 Arg Trp Leu Ser Thr 530 GTC TAT GCT GGC
AGT AAT CTG GCC ATG CAG GAC 1986 ACC AGC CGC CGA CCC Val Tyr Ala
Gly Ser Asn Leu Ala Met Gln Asp 535 540 545 Thr Ser Arg Arg Pro 550
TGC CAC AGC CAG GAC TTC TCC GTG CAC GGC AAC 2034 ATC ATC AAC GGG
GCT Cys His Ser Gln Asp Phe Ser Val His Gly Asn 555 560 Ile Ile Asn
Gly Ala 565 GAC TGG CAC ACG GTC CCC GGG AGC ATG AAT GAC 2082 TTC
AGC TAC CTA CAC Asp Trp His Thr Val Pro Gly Ser Met Asn Asp 570 575
Phe Ser Tyr Leu His 580 ACC AAC TGC TTT GAG GTC ACT GTG GAG CTG TCC
2130 TGT GAC AAG TTC CCT Thr Asn Cys Phe Glu Val Thr Val Glu Leu
Ser 585 590 Cys Asp Lys Phe Pro 595 CAC GAG AAT GAA TTG CCC CAG GAG
TGG GAG AAC 2178 AAC AAA GAC GCC CTC His Glu Asn Glu Leu Pro Gln
Glu Trp Glu Asn 600 605 Asn Lys Asp Ala Leu 610 CTC ACC TAC CTG GAG
CAG GTG CGC ATG GGC ATT 2226 GCA GGA GTG GTG AGG Leu Thr Tyr Leu
Glu Gln Val Arg Met Gly Ile 615 620 625 Ala Gly Val Val Arg 630 GAC
AAG GAC ACG GAG CTT GGG ATT GCT GAC GCT 2274 GTC ATT GCC GTG GAT
Asp Lys Asp Thr Glu Leu Gly Ile Ala Asp Ala 635 640 Val Ile Ala Val
Asp 645 GGG ATT AAC CAT GAC GTG ACC ACG GCG TGG GGC 2322 GGG GAT
TAT TGG CGT Gly Ile Asn His Asp Val Thr Thr Ala Trp Gly 650 655 Gly
Asp Tyr Trp Arg 660 CTG CTG ACC CCA GGG GAC TAC ATG GTG ACT GCC
2370 AGT GCC GAG GGC TAC Leu Leu Thr Pro Gly Asp Tyr Met Val Thr
Ala 665 670 Ser Ala Glu Gly Tyr 675 CAT TCA GTG ACA CGG AAC TGT CGG
GTC ACC TTT 2418 GAA GAG GGC CCC TTC His Ser Val Thr Arg Asn Cys
Arg Val Thr Phe 680 685 Glu Glu Gly Pro Phe 690 CCC TGC AAT TTC GTG
CTC ACC AAG ACT CCC AAA 2466 CAG AGG CTG CGC GAG Pro Cys Asn Phe
Val Leu Thr Lys Thr Pro Lys 695 700 705 Gln Arg Leu Arg Glu 710 CTG
CTG GCA GCT GGG GCC AAG GTG CCC CCG GAC 2514 CTT CGC AGG CGC CTG
Leu Leu Ala Ala Gly Ala Lys Val Pro Pro Asp 715 720 Leu Arg Arg Arg
Leu 725 GAG CGG CTA AGG GGA CAG AAG GAT TGA 2561 TACCTGCGGT
TTAAGAGCCC Glu Arg Leu Arg Gly Gln Lys Asp * 730 735 TAGGGCAGGC
TGGACCTGTC AAGACGGGAA GGGGAAGAGT 2621 AGAGAGGGAG GGACAAAGTG
AGGAAAAGGT GCTCATTAAA GCTACCGGGC ACCTTAAAAA 2681 AAAAAAAAAA
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAGGG CGCCCGCT 2719 TABLE 2
Human FDH02 nucleotide and predicted amino acid sequence. SEQ ID
NO: 3 and 4. The predicted signal sequence is underlined. The
mature peptide should begin about at Asp (position 21). CAGGTACCGG
TCCGGAATTC CCGGGTCGAC CCACGCGTCC 60 GGTTTGGTGT GAGGCTGCGA
GCCGCCGCGA GTTCTCACGG TCCCGCCGGC GCCACCACCG 120 CGGTCACTCA
CCGCCGCCGC CGCCACCACT GCCACCACGG TCGCCTGCCA CAGGTGTCTG 180
CAATTGAACT CCAAGGTGCA GA ATG GTT TGG AAA GTA GCT GTA TTC CTC AGT
227 GTG GCC CTG GGC ATT Met Val Trp Lys Val Ala Val Phe Leu Ser 1 5
10 Val Ala Leu Gly Ile 15 GGT GCC GTT CCT ATA GAT GAT CCT GAA GAT
GGA 275 GGC AAG CAC TGG GTG Gly Ala Val Pro Ile Asp Asp Pro Glu Asp
Gly 20 25 Gly Lys His Trp Val 30 GTG ATC GTG GCA GGT TCA AAT GGC
TGG TAT AAT 323 TAT AGG CAC CAG GCA Val Ile Val Ala Gly Ser Asn Gly
Trp Tyr Asn 35 40 Tyr Arg His Gln Ala 45 GAC GCG TGC CAT GCC TAC
CAG ATC ATT CAC CGC 371 AAT GGG ATT CCT GAC Asp Ala Cys His Ala Tyr
Gln Ile Ile His Arg 50 55 Asn Gly Ile Pro Asp 60 GAA CAG ATC GTT
GTG ATG ATG TAC GAT GAC ATT 419 GCT TAC TCT GAA GAC Glu Gln Ile Val
Val Met Met Tyr Asp Asp Ile 65 70 Ala Tyr Ser Glu Asp 75 AAT CCC
ACT CCA GGA ATT GTG ATC AAC AGG CCC 467 AAT GGC ACA GAT GTC Asn Pro
Thr Pro Gly Ile Val Ile Asn Arg Pro 80 85 90 Asn Gly Thr Asp Val 95
TAT CAG GGA GTC CCG AAG GAC TAC ACT GGA GAG 515 GAT GTT ACC CCA CAA
Tyr Gln Gly Val Pro Lys Asp Tyr Thr Gly Glu 100 105 Asp Val Thr Pro
Gln 110 AAT TTC CTT GCT GTG TTG AGA GGC GAT GCA GAA 563 GCA GTG AAG
GGC ATA Asn Phe Leu Ala Val Leu Arg Gly Asp Ala Glu 115 120 Ala Val
Lys Gly Ile 125 GGA TCC GGC AAA GTC CTG AAG AGT GGC CCC CAG 611 GAT
CAC GTG TTC ATT Gly Ser Gly Lys Val Leu Lys Ser Gly Pro Gln 130 135
Asp His Val Phe Ile 140 TAC TTC ACT GAC CAT GGA TCT ACT GGA ATA CTG
659 GTT TTT CCC AAT GAA Tyr Phe Thr Asp His Gly Ser Thr Gly Ile Leu
145 150 Val Phe Pro Asn Glu 155 GAT CTT CAT GTA AAG GAC CTG AAT GAG
ACC ATC 707 CAT TAC ATG TAC AAA Asp Leu His Val Lys Asp Leu Asn Glu
Thr Ile 160 165 170 His Tyr Met Tyr Lys 175 CAC AAA ATG TAC CGA AAG
ATG GTG TTC TAC ATT 755 GAA GCC TGT GAG TCT His Lys Met Tyr Arg Lys
Met Val Phe Tyr Ile 180 185 Glu Ala Cys Glu Ser 190 GGG TCC ATG ATG
AAC CAC CTG CCG GAT AAC ATC 803 AAT GTT TAT GCA ACT Gly Ser Met Met
Asn His Leu Pro Asp Asn Ile 195 200 Asn Val Tyr Ala Thr 205 ACT GCT
GCC AAC CCC AGA GAG TCG TCC TAC GCC 851 TGT TAC TAT GAT GAG Thr Ala
Ala Asn Pro Arg Glu Ser Ser Tyr Ala 210 215 Cys Tyr Tyr Asp Glu 220
AAG AGG TCC ACG TAC CTG GGG GAC TGG TAC AGC 899 GTC AAC TGG ATG GAA
Lys Arg Ser Thr Tyr Leu Gly Asp Trp Tyr Ser 225 230 Val Asn Trp Met
Glu 235 GAC TCG GAC GTG GAA GAT CTG ACT AAA GAG ACC 947 CTG CAC AAG
CAG TAC Asp Ser Asp Val Glu Asp Leu Thr Lys Glu Thr 240 245 250 Leu
His Lys Gln Tyr 255 CAC CTG GTA AAA TCG CAC ACC AAC ACC AGC CAC 995
GTC ATG CAG TAT GGA His Leu Val Lys Ser His Thr Asn Thr Ser His 260
265 Val Met Gln Tyr Gly 270 AAC AAA ACA ATC TCC ACC ATG AAA GTG ATG
CAG 1043 TTT CAG GGT ATG AAA Asn Lys Thr Ile Ser Thr Met Lys Val
Met Gln 275 280 Phe Gln Gly Met Lys 285 CGC AAA GCC AGT TCT CCC GTC
CCC CTA CCT CCA 1091 GTC ACA CAC CTT GAC Arg Lys Ala Ser Ser Pro
Val Pro Leu Pro Pro 290 295 Val Thr His Leu Asp 300 CTC ACC CCC AGC
CCT GAT GTG CCT CTC ACC ATC 1139 ATG AAA AGG AAA CTG Leu Thr Pro
Ser Pro Asp Val Pro Leu Thr Ile 305 310 Met Lys Arg Lys Leu 315 ATG
AAC ACC AAT GAT CTG GAG GAG TCC AGG CAG 1187 CTC ACG GAG GAG ATC
Met Asn Thr Asn Asp Leu Glu Glu Ser Arg Gln 320 325 330 Leu Thr Glu
Glu Ile 335 CAG CGG CAT CTG GAT GCC AGG CAC CTC ATT GAG 1235 AAG
TCA GTG CGT AAG Gln Arg His Leu Asp Ala Arg His Leu Ile Glu 340 345
Lys Ser Val Arg Lys 350 ATC GTC TCC TTG CTG GCA GCG TCC GAG GCT GAG
1283 GTG GAG CAG CTC CTG Ile Val Ser Leu Leu Ala Ala Ser Glu Ala
Glu 355 360 Val Glu Gln Leu Leu 365 TCC GAG AGA GCC CCG CTC ACG GGG
CAC AGC TGC 1331 TAC CCA GAG GCC CTG Ser Glu Arg Ala Pro Leu Thr
Gly His Ser Cys 370 375 Tyr Pro Glu Ala Leu 380 CTG CAC TTC CGG ACC
CAC TGC TTC AAC TGG CAC 1379 TCC CCC ACG TAC GAG Leu His Phe Arg
Thr His Cys Phe Asn Trp His 385 390 Ser Pro Thr Tyr Glu 395 TAT
GCG TTG AGA CAT TTG TAC GTG CTG GTC AAC 1427 CTT TGT GAG AAG CCG
Tyr Ala Leu Arg His Leu Tyr Val Leu Val Asn 400 405 410 Leu Cys Glu
Lys Pro 415 TAT CCA CTT CAC AGG ATA AAA TTG TCC ATG GAC 1475 CAC
GTG TGC CTT GGT Tyr Pro Leu His Arg Ile Lys Leu Ser Met Asp 420 425
His Val Cys Leu Gly 430 CAC TAC TGA AGAGCTGCCT CCTGGAAGCT
TTTCCAAGTG 1524 TGAGCGCCCC His Tyr * CCCGACTGTG TGCTGATCAG
AGACTGGAGA GGTGGAGTGA 1584 GAAGTCTCCG CTGCTCGGGC CCTCCTGGGG
AGCCCCCGCT CCAGGGCTCG CTCCAGGACC 1644 TTCTTCACAA GATGACTTGC
TCGCTGTTAC CTGCTTCCCC AGTCTTTTCT GAAAAACTAC 1704 AAATTAGGGT
GGGAAAAGCT CTGTATTGAG AAGGGTCATA TTTGCTTTCT AGGAGGTTTG 1764
TTGTTTTGCC TGTTAGTTTT GAGGAGCAGG AAGCTCATGG GGGCTTCTGT AGCCCCTCTC
1824 AAAAGGAGTC TTTATTCTGA GAATTTGAAG CTGAAACCTC TTTAAATTTT
CAGAATGATT 1884 TTATTGAAGA GGGCCGCAAG CCCCAAATGG AAAACTGTTT
TTAGAAAATA TGATGATTTT 1944 TGATTGCTTT TGTATTTAAT TCTGCAGGTG
TTCAAGTCTT AAAAAATAAA GATTTATAAC 2004 AGAACCCAAA AAAAAAAAAA
AAAAAAAAAA AAAAAAGGGC GGCCGC 2030
[0015]
3TABLE 3 Human D1B2 (MS2) partial nucleic acid and predicted amino
acid sequence. SEQ ID NO: 5 and 6. The predicted signal sequence is
under- lined. The mature peptide should begin about at Ser
(position 20). ATG CGC GGT CTC GGG CTC TGG CTG CTG GGC GCG 48 ATG
ATG CTG CCT GCG Met Arg Gly Leu Gly Leu Trp Leu Leu Gly Ala 1 5 10
Met Met Leu Pro Ala 15 ATT GCC CCC AGC CGG CCC TGG GCC CTC ATG GAG
96 CAG TAT GAG GTC GTG Ile Ala Pro Ser Arg Pro Trp Ala Leu Met Glu
20 25 Gln Tyr Glu Val Val 30 TTG CCG CGG TGT CTG CCA GGC CCC CGA
GTC CGC 144 CGA GCT CTG CCC TCC Leu Pro Arg Cys Leu Pro Gly Pro Arg
Val Arg 35 40 Arg Ala Leu Pro Ser 45 CAC TTG GGC CTG CAC CCA GAG
AGG GTG AGC TAC 192 GTC CTT GGG GCC ACA His Leu Gly Leu His Pro Glu
Arg Val Ser Tyr 50 55 Val Leu Gly Ala Thr 60 GGG CAC AAC TTC ACC
CTC CAC CTG CGG AAG AAC 240 AGG GAC CTG CTG GGT Gly His Asn Phe Thr
Leu His Leu Arg Lys Asn 65 70 75 Arg Asp Leu Leu Gly 80 TCC GGC TAC
ACA GAG ACC TAT ACG GCT GCC AAT 288 GGC TCC GAG GTG ACG Ser Gly Tyr
Thr Glu Thr Tyr Thr Ala Ala Asn 85 90 Gly Ser Glu Val Thr 95 GAG
CAG CCT CGC GGG CAG GAC CAC TGC TTC TAC 336 CAG GGC CAC GTA GAG Glu
Gln Pro Arg Gly Gln Asp His Cys Phe Tyr 100 105 Gln Gly His Val Glu
110 GGG TAC CCG GAC TCA GCC GCC AGC CTC AGC ACC 384 TGT GCC GGC CTC
AGG Gly Tyr Pro Asp Ser Ala Ala Ser Leu Ser Thr 115 120 Cys Ala Gly
Leu Arg 125 GGT TTC TTC CAG GTG GGG TCA GAC CTG CAC CTG 432 ATC GAG
CCC CTG GAT Gly Phe Phe Gln Val Gly Ser Asp Leu His Leu 130 135 Ile
Glu Pro Leu Asp 140 GAA GGT GGC GAG GGC GGA CGG CAC GCC GTG TAC 480
CAG GCT GAG CAC CTG Glu Gly Gly Glu Gly Gly Arg His Ala Val Tyr 145
150 155 Gln Ala Glu His Leu 160 CTG CAG ACG GCC GGG ACC TGC GGG GTC
AGC GAC 528 GAC AGC CTG GGC AGC Leu Gln Thr Ala Gly Thr Cys Gly Val
Ser Asp 165 170 Asp Ser Leu Gly Ser 175 CTC CTG GGA CCC CGG ACG GCA
GCC GTC TTC AGG 576 CCT CGG CCC GGG GAC Leu Leu Gly Pro Arg Thr Ala
Ala Val Phe Arg 180 185 Pro Arg Pro Gly Asp 190 TCT CTG CCA TCC CGA
GAG ACC CGC TAC GTG GAG 624 CTG TAT GTG GTC GTG Ser Leu Pro Ser Arg
Glu Thr Arg Tyr Val Glu 195 200 Leu Tyr Val Val Val 205 GAC AAT GCA
GAG TTC CAG ATG CTG GGG AGC GAA 672 GCA GCC GTG CGT CAT Asp Asn Ala
Glu Phe Gln Met Leu Gly Ser Glu 210 215 Ala Ala Val Arg His 220 CGG
GTG CTG GAG GTG GTG AAT CAC GTG GAC AAG 720 CTA TAT CAG AAA CTC Arg
Val Leu Glu Val Val Asn His Val Asp Lys 225 230 235 Leu Tyr Gln Lys
Leu 240 AAC TTC CGT GTG GTC CTG GTG GGC CTG GAG ATT 768 TGG AAT AGT
CAG GAC Asn Phe Arg Val Val Leu Val Gly Leu Glu Ile 245 250 Trp Asn
Ser Gln Asp 255 AGG TTC CAC GTC AGC CCC GAC CCC AGT GTC ACA 816 CTG
GAG AAC CTC CTG Arg Phe His Val Ser Pro Asp Pro Ser Val Thr 260 265
Leu Glu Asn Leu Leu 270 ACC TGG CAG GCA CGG CAA CGG ACA CGG CGG CAC
864 CTG CAT GAC AAC GTA Thr Trp Gln Ala Arg Gln Arg Thr Arg Arg His
275 280 Leu His Asp Asn Val 285 CAG CTC ATC ACG GGT GTC GAC TTC ACC
GGG ACT 912 ACT GTG GGG TTT GCC Gln Leu Ile Thr Gly Val Asp Phe Thr
Gly Thr 290 295 Thr Val Gly Phe Ala 300 AGG GTG TCC GCC ATG TGC TCC
CAC AGC TCA GGG 960 GCT GTG AAC CAG GAC Arg Val Ser Ala Met Cys Ser
His Ser Ser Gly 305 310 315 Ala Val Asn Gln Asp 320 CAC AGC AAG AAC
CCC GTG GGC GTG GCT GCA CCA 1008 TGG CCC ATG AGA TGG His Ser Lys
Asn Pro Val Gly Val Ala Ala Pro 325 330 Trp Pro Met Arg Trp 335 GCC
ACA ACC TGG GCA TGG ACC ATG ATG AGA ACG 1056 TCC AGG GCT GCC GCT
Ala Thr Thr Trp Ala Trp Thr Met Met Arg Thr 340 345 Ser Arg Ala Ala
Ala 350 GCC AGG AAC GCT TCG AGG CCG GCC GCT GCA TCA 1104 TGG CAG
GCA GCA TTG Ala Arg Asn Ala Ser Arg Pro Ala Ala Ala Ser 355 360 Trp
Gln Ala Ala Leu 365 GCT CCA GTT TCC CCA GGA TGT TCA GTG ACT GCA
1152 GCC AGG CCT ACC TGG Ala Pro Val Ser Pro Gly Cys Ser Val Thr
Ala 370 375 Ala Arg Pro Thr Trp 380 AGA GCT TTT TGG AGC GGC CGC
1173 Arg Ala Phe Trp Ser Oly Arg 385 390
[0016] The proteases of this invention are defined in part by their
sequences, and by their physicochemical and biological properties.
The biological properties of the human proteases described herein,
e.g., human APG04, D1B2, and GFDO2, are defined by their amino acid
sequences, and mature sizes. They also should share certain
biological enzymatic properties.
[0017] The human protease APG04 is a carboxypeptidase H domain
containing protein isolated from CD1a+ CD34+ dendritic cells. This
protein family includes soluble enzymes which process hormones from
precursors. APG04 shares amino acid homology to aebp1, a soluble
trancriptional repressor, a bone-related carboxypeptidase 2', and
other carboxypeptidases. See, e.g., Manser, et al. (1990) Biochem.
J. 267:517-525; He, et al. (1995) Nature 378:92-96; and Jung, et
al. (1991) Mol. Endocrinol. 5:1257-1268. Known physiological
substrates for this family of carboxypeptidases include, e.g.,
enkephalin and bradykinin. APG04 does not appear to contain a
transmembrane domain. Expression analysis found this protease to be
a late activation marker in dendritic cells. Signal was also
detected in U937 monocytic cells and B cells, suggesting a
physiological role in immmune function.
[0018] FHD02, also isolated from dendritic cells, exhibits amino
acid homology to several hemaglobinases of some parasites and
proteases from various seeds or fruits. See, e.g., Klinkert, et al.
(1989) Mol. Biocyhem. Parasitol. 33:113-122; el Meanawy, et al.
(1990) Am. J. Trop. Med. Hyg. 43:67-78; Davis, et al. (1987) J.
Biol. Chem. 262:12851-12858; and Becker, et al. (1995) Eur. J.
Biochem. 228:456-462. Substrates for a related protease,
asparaginyl endopeptidase, are described in Abe, et al. (1993) J.
Biol. Chem. 268:3525-3429, which also are prime candidates of
substrates for FDH02. FHD02 shares significant nucleic acid
homology with an EST of unidentified function, from GenBank,
designated emb.vertline.F01300.vertline.HSBC6B022. In addition to
dendritic cells, FHD02 is expressed in placenta, spleen, small
intestine, and monocytes treated with LPS and IFN-.gamma.. D1B2 is
the human homolog of a mouse antigen designated mouse MS2. The
extracellular region contains a clear metalloproteinase domain
related to a family of several well characterized snake venom
proteins which seem to inhibit blood clotting processes, e.g.,
Jararhagin precursor. See, e.g., Gomis-Rueth, et al. (1994) J. Mol.
Biol. 239:513-544; Yoshida, et al. (1990) Int'l. Immunol.
2:585-591; Takeya, et al. (1989) J. Biochem 106:151-157; and de
Araujo, et al. (1995) Arch. Biochem. Biophys. 320:141-148. The
similarity in sequence comparison with the snake venom proteins
extends beyond the protease activity domain, implying a second
functional domain in the protein, called the disintegin domain, a
platelet aggregation inhibitor. See, e.g., Katagiri, et al. (1995)
Cytoaenet. Cell Genet. 68:39-44; and Emi, et al. (1993) Nat. Genet.
5:151-157. Based on the strong structural homology with these snake
venom proteins, likely substrates for D1B2 may include, e.g.,
insulin, type IV collagen, or fibrinogen.
[0019] D1B2 was initially found by subtraction of a resting
dendritic cell library and a monocyte library. The full length
clone was isolated from a 70% CD1a+ library (CD34+ hematopoietic
progenitor cells cultured for 12 days in GM-CSF and TNF.alpha.).
D1B2 shares approximately 68% identity with mouse MS2. Mouse MS2 is
expressed mainly in macrophages, whereas D1B2 is expressed mainly
in monocytes, resting and activated dendritic cells, resting Th1
cells, activated T cells, activated PBLs, and activated spleen
cells.
[0020] One of skill will readily recognize that some sequence
variations may be tolerated, e.g., conservative substitutions or
positions remote from the critical helical structures and remote
from the-identified critial active site regions, without altering
significantly the biological activity of each respective
molecule.
[0021] APG04, FDH02, or D1B2 proteins are present in specific cell
types, e.g., dendritic cells, and the interaction of the protease
with a substrate will be important for mediating various aspects of
cellular physiology or development. The cellular types which
express messages encoding APG02, FDH02, and D1B2 suggest that
signals important in cell differentiation and development are
mediated by them. See, e.g., Gilbert (1991) Developmental Biology
(3d ed.) Sinauer Associates, Sunderland, Mass.; Browder, et al.
(1991) Developmental Biology (3d ed.) Saunders, Philadelphia, Pa.;
Russo, et al. (1992) Development: The Molecular Genetic Approach
Springer-Verlag, New York, N.Y.; and Wilkins (1993) Genetic
Analysis of Animal Development (2d ed.) Wiley-Liss, New York, N.Y.
In particular, the proteases may be necessary for the conversion of
pro-proteins to proteins, e.g., cytokine or protein precursors to
mature forms, or for proper immunological function, e.g., antigen
processing and presentation.
[0022] II. Definitions
[0023] The term "binding composition" refers to molecules that bind
with specificity to APG04, FDH02, or D1B2, respectively, e.g., in
an antibody-antigen interaction. However, other compounds, e.g.,
receptor proteins, may also specifically associate with APG04,
FDH02, or D1B2 to the exclusion of other molecules. Typically, the
association will be in a natural physiologically relevant
protein-protein interaction, either covalent or non-covalent, and
may include members of a multiprotein complex, including carrier
compounds or dimerization partners. The molecule may be a polymer,
or chemical reagent. A functional analog may be a protease with
structural modifications, or may be a wholly unrelated molecule,
e.g., which has a molecular shape which interacts with the
appropriate substrate cleavage determinants.
[0024] The term "binding agent:APG04, FDH02 or D1B2 protein
complex", as used herein, refers to a complex of a binding agent
and an APG04, FDH02, or D1B2 protein that is formed by specific
binding of the binding agent to the APG04, FDH02, or D1B2 protein.
Specific binding of the binding agent means that the binding agent
has a specific binding site that recognizes a site on the APG04,
FDH02, or D1B2 protein. For example, antibodies raised to an APG04,
FDH02, or D1B2 protein and recognizing an epitope on the APG04,
FDH02, or D1B2 protein are capable of forming a binding
agent:APG04, FDH02, or D1B2 protein complex by specific binding.
Typically, the formation of a binding agent: APG04, FDH02, or D1B2
protein complex allows the measurement of APG04, FDH02, or D1B2
protein in a mixture of other proteins and biologics. The term
"antibody:APG04, FDH02, or D1B2 protein complex" refers to an
embodiment in which the binding agent is an antibody. The antibody
may be monoclonal, polyclonal, or a binding fragment of an
antibody, e.g, an Fab of F(ab)2 fragment. The antibody will
preferably be a polyclonal antibody for cross-reactivity
determinations.
[0025] "Homologous" nucleic acid sequences, when compared, exhibit
significant similarity or identity. The standards for homology in
nucleic acids are either measures for homology generally used in
the art by sequence comparison and/or phylogenetic relationship, or
based upon hybridization conditions. Hybridization conditions are
described in greater detail below.
[0026] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other biologic components which naturally accompany a native
sequence, e.g., proteins and flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs, or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. An isolated nucleic acid will
usually contain homogeneous nucleic acid molecules, but will, in
some embodiments, contain nucleic acids with minor sequence
heterogeneity. This heterogeneity is typically found at the polymer
ends or portions not critical to a desired biological function or
activity.
[0027] As used herein, the term "APG04, FDH02, or D1B2 protein"
shall encompass, when used in a protein context, a protein having
amino acid sequences, particularly from the protein motif portions,
shown in SEQ ID NO: 2, 4, or 6. In many contexts, a significant
fragment of such a protein will be functionally equivalent. The
invention also embraces a polypeptide which exhibits similar
structure to human APG04, FDH02, or D1B2 protein, e.g., which
interacts with APG04, FDH02, or D1B2 specific binding components.
These binding components, e.g., antibodies, typically bind to
APG04, FDH02, or D1B2 protein with high affinity, e.g., at least
about 100 nM, usually better than about 30 nM, preferably better
than about 10 nM, and more preferably at better than about 3
nM.
[0028] The term "polypeptide" or "protein" as used herein includes
a significant fragment or segment of protease motif portion of
APG04, FDH02, or D1B2 protein, and encompasses a stretch of amino
acid residues of at least about 8 amino acids, generally at least
about 10 amino acids, more generally at least about 12 amino acids,
often at least about 14 amino acids, more often at least about 16
amino acids, typically at least about 18 amino acids, more
typically at least about 20 amino acids, usually at least about 22
amino acids, more usually at least about 24 amino acids, preferably
at least about 26 amino acids,more preferably at least about 28
amino acids, and, in particularly preferred embodiments, at least
about 30 or more amino acids, e.g., 35, 40, 45, 50, 60, 70, 80,
etc.
[0029] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not-naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus, for
example, products made by transforming cells with any non-naturally
occurring vector is encompassed, as are nucleic acids comprising
sequence derived using any synthetic oligonucleotide process. Such
is often done to replace a codon with a redundant codon encoding
the same or a conservative amino acid, while typically introducing
or removing a sequence recognition site. Alternatively, it is
performed to join together nucleic acid segments of desired
functions to generate a single genetic entity comprising a desired
combination of functions not found in the commonly available
natural forms. Restriction enzyme recognition sites are often the
target of such artificial manipulations, but other site specific
targets, e.g., promoters, DNA replication sites, regulation
sequences, control sequences, or other useful features may be
incorporated by design. A similar concept is intended for a
recombinant, e.g., fusion, polypeptide. Specifically included are
synthetic nucleic acids which, by genetic code redundancy, encode
polypeptides similar to fragments of these antigens, and fusions of
sequences from various different species variants.
[0030] "Solubility" is reflected by sedimentation measured in
Svedberg units, which are a measure of the sedimentation velocity
of a molecule under particular conditions. The determination of the
sedimentation velocity was classically performed in an analytical
ultracentrifuge, but is typically now performed in a standard
ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d
ed.) W. H. Freeman & Co., San Francisco, Calif.; and Cantor and
Schimmel (1980) Biophysical Chemistry parts 1-3, W. H. Freeman
& Co., San Francisco, Calif. As a crude determination, a sample
containing a putatively soluble polypeptide is spun in a standard
full sized ultracentrifuge at about 50K rpm for about 10 minutes,
and soluble molecules will remain in the supernatant. A soluble
particle or polypeptide will typically be less than about 30 S,
more typically less than about 15 S, usually less than about 10 S,
more usually less than about 6 S, and, in particular embodiments,
preferably less than about 4 S, and more preferably less than about
3 S. Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. and more
usually greater than about 22.degree. C. For diagnostic purposes,
the temperature will usually be about room temperature or warmer,
but less than the denaturation temperature of components in the
assay. For therapeutic purposes, the temperature will usually be
body temperature, typically about 37.degree. C. for humans, though
under certain situations the temperature may be raised or lowered
in situ or in vitro.
[0031] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents in a manner which approximates natural
lipid bilayer interactions.
[0032] The solvent will usually be a biologically compatible
buffer, of a type used for preservation of biological activities,
and will usually approximate a physiological solvent. Usually the
solvent will have a neutral pH, typically between about 5 and 10,
and preferably about 7.5. On some occasions, a detergent will be
added, typically a mild non-denaturing one, e.g., CHS (cholesteryl
hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein.
[0033] "Substantially pure" in a protein context typically means
that the protein is isolated from other contaminating proteins,
nucleic acids, and other biologicals derived from the original
source organism. Purity, or "isolation" may be assayed by standard
methods, and will ordinarily be at least about 50% pure, more
ordinarily at least about 60% pure, generally at least about 70%
pure, more generally at least about 80% pure, often at least about
85% pure, more often at least about 90% pure, preferably at least
about 95% pure, more preferably at least about 98% pure, and in
most preferred embodiments, at least 99% pure. Similar concepts
apply, e.g., to antibodies or nucleic acids.
[0034] "Substantial similarity" in the nucleic acid sequence
comparison context means either that the segments, or their
complementary strands, when compared, are identical when optimally
aligned, with appropriate nucleotide insertions or deletions, in at
least about 50% of the nucleotides, generally at least about 56%,
more generally at least about 59%, ordinarily at least about 62%,
more ordinarily at least about 65%, often at least about 68%, more
often at least about 71%, typically at least about 74%, more
typically at least about 77%, usually at least about 80%, more
usually at least about 85%, preferably at least about 90%, more
preferably at least about 95 to 98% or more, and in particular
embodiments, as high at about 99% or more of the nucleotides.
Alternatively, substantial similarity exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence derived from SEQ ID NO:
1, 3, or 5. Typically, selective hybridization will occur when
there is at least about 55% similarity over a stretch of at least
about 30 nucleotides, preferably at least about 65% over a stretch
of at least about 25 nucleotides, more preferably at least about
75%, and most preferably at least about 90% over about 20
nucleotides. See Kanehisa (1984) Nuc. Acids Res. 12:203-213. The
length of similarity comparison, as described, may be over longer
stretches, and in certain embodiments will be over a stretch of at
least about 17 nucleotides, usually at least about 20 nucleotides,
more usually at least about 24 nucleotides, typically at least
about 28 nucleotides, more typically at least-about 40 nucleotides,
preferably at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides, e.g., 150, 200, etc.
[0035] "Stringent conditions", in referring to homology or
substantial similarity in the hybridization context, will be
stringent combined conditions of salt, temperature, organic
solvents, and other parameters, typically those controlled in
hybridization reactions. The combination of parameters is more
important than the measure of any single parameter. See, e.g.,
Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. A nucleic acid
probe which binds to a target nucleic acid under stringent
conditions is specific for said target nucleic acid. Such a probe
is typically more than 11 nucleotides in length, and is
sufficiently identical or complementary to a target nucleic acid
over the region specified by the sequence of the probe to bind the
target under stringent hybridization conditions.
[0036] APG04, FDH02, or D1B2 proteins from other mammalian species
can be cloned and isolated by cross-species hybridization of
closely related species. See, e.g., below. Similarity may be
relatively low between distantly related species, and thus
hybridization of relatively closely related species is advisable.
Alternatively, preparation of an antibody preparation which
exhibits less species specificity may be useful in expression
cloning approaches.
[0037] The phrase "specifically binds to an antibody" or
"specifically immunoreactive with"; when referring to a protein or
peptide, refers to a binding reaction which is determinative of the
presence of the protein in the presence of a heterogeneous
population of proteins and other biological components. Thus, under
designated immunoassay conditions, the specified antibodies bind to
a particular protein and do not significantly bind other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, antibodies
raised to the human APG04, FDH02, or D1B2 protein immunogen with
the amino acid sequence depicted in SEQ ID NO: 2, 4, or 6 can be
selected by immunoaffinity or similar methods to obtain antibodies
specifically immunoreactive with APG04, FDH02, or D1B2 proteins and
not with other proteins.
[0038] III. Nucleic Acids
[0039] APG04, FDH02, or D1B2 proteins are exemplary of a larger
class of structurally and functionally related proteins. These
proteins will serve to cleave various proteins produced or
processed by various cell types. The preferred embodiments, as
disclosed, will be useful in standard procedures to isolate genes
from different individuals or other species, e.g., warm blooded
animals, such as birds and mammals. Cross hybridization will allow
isolation of related genes encoding proteins from individuals,
strains, or species. A number of different approaches are available
to successfully isolate a suitable nucleic acid clone based upon
the information provided herein. Southern blot hybridization
studies can qualitatively determine the presence of homologous
genes in human, monkey, rat, dog, cow, and rabbit genomes under
specific hybridization conditions.
[0040] Complementary sequences will also be used as probes or
primers. Based upon identification of the likely amino terminus,
other peptides should be particularly useful, e.g., coupled with
anchored vector or poly-A complementary PCR techniques or with
complementary DNA of other peptides.
[0041] Techniques for nucleic acid manipulation of genes encoding
APG04, FDH02, or D1B2 proteins, such as subcloning nucleic acid
sequences encoding polypeptides into expression vectors, labelling
probes, DNA hybridization, and the like are described generally in
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd
ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, N.Y., which is incorporated herein by reference. This manual
is hereinafter referred to as "Sambrook, et al.".
[0042] There are various methods of isolating DNA sequences
encoding APG04, FDH02, or D1B2 proteins. For example, DNA is
isolated from a genomic or cDNA library using labeled
oligonucleotide probes having sequences identical or complementary
to the sequences disclosed herein. Full-length probes may be used,
or oligonucleotide probes may be generated by comparison of the
sequences disclosed. Such probes can be used directly in
hybridization assays to isolate DNA encoding APG04, FDH02, or D1B2
proteins, or probes can be designed for use in amplification
techniques such as PCR, for the isolation of DNA encoding APG04,
FDH02, or D1B2 proteins.
[0043] To prepare a cDNA library, mRNA is isolated from cells which
expresses an APG04, FDH02, or D1B2 protein. cDNA is prepared from
the mRNA and ligated into a recombinant vector. The vector is
transfected into a recombinant host for propagation, screening, and
cloning. Methods for making and screening cDNA libraries are well
known. See Gubler and Hoffman (1983) Gene 25:263-269 and Sambrook,
et al.
[0044] For a genomic library, the DNA can be extracted from tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation and cloned in bacteriophage lambda vectors.
These vectors and phage are packaged in vitro, as described in
Sambrook, et al. Recombinant phage are analyzed by plaque
hybridization as described in Benton and Davis (1977) Science
196:180-182. Colony hybridization is carried out as generally
described in e.g., Grunstein, et al. (1975) Proc. Natl. Acad. Sci.
USA. 72:3961-3965.
[0045] DNA encoding an APG04, FDH02, or D1B2 protein can be
identified in either cDNA or genomic libraries by its ability to
hybridize with the nucleic acid probes described herein, e.g., in
colony or plaque hybridization assays. The corresponding DNA
regions are isolated by standard methods familiar to those of skill
in the art. See, e.g., Sambrook, et al.
[0046] Various methods of amplifying target sequences, such as the
polymerase chain reaction, can also be used to prepare DNA encoding
APG04, FDH02, or D1B2 proteins. Polymerase chain reaction (PCR)
technology is used to amplify such nucleic acid sequences directly
from mRNA, from cDNA, and from genomic libraries or cDNA libraries.
The isolated sequences encoding APG04, FDH02, or D1B2 proteins may
also be used as templates for PCR amplification.
[0047] Typically, in PCR techniques, oligonucleotide primers
complementary to two 5' regions in the DNA region to be amplified
are synthesized. The polymerase chain reaction is then carried out
using the two primers. See Innis, et al. (eds.) (1990) PCR
Protocols: A Guide to Methods and Applications Academic Press, San
Diego, Calif. Primers can be selected to amplify the entire regions
encoding a full-length human APG04, FDH02, or D1B2 protein or to
amplify smaller DNA segments as desired. Once such regions are
PCR-amplified, they can be sequenced and oligonucleotide probes can
be prepared from sequence obtained using standard techniques. These
probes can then be used to isolate DNA's encoding APG04, FDH02, or
D1B2 proteins.
[0048] Oligonucleotides for use as probes are usually chemically
synthesized according to the solid phase phosphoramidite triester
method first described by Beaucage and Carruthers (1983)
Tetrahedron Lett. 22(20):1859-1862, or using an automated
synthesizer, as described in Needham-VanDevanter, et al. (1984)
Nucleic Acids Res, 12:6159-6168. Purification of oligonucleotides
is performed e.g., by native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson and Regnier (1983) J.
Chrom. 255:137-149. The sequence of the synthetic oligonucleotide
can be verified using, e.g., the chemical degradation method of
Maxam, A. M. and Gilbert, W. in Grossman, L. and Moldave (eds.)
(1980) Methods in Enzymology 65:499-560 Academic Press, New
York.
[0049] An isolated nucleic acid encoding a human APG04, FDH02, or
D1B2 protein was identified. The nucleotide sequence, corresponding
open reading frames, and mature peptides are provided in Tables 1,
2, or 3 and SEQ ID NO: 1-6.
[0050] This invention provides isolated DNA or fragments to encode
an APG04, FDH02, or D1B2 protein or specific fragment thereof.
[0051] In addition, this invention provides isolated or recombinant
DNA which encodes a protein or polypeptide, and which is capable of
hybridizing under appropriate conditions, e.g., high stringency,
with the DNA sequences described herein. Said biologically active
protein or polypeptide can be a functional protease segment, or
fragment, and have an amino acid sequence as disclosed in SEQ ID
NO: 2, 4, or 6. Preferred embodiments will be full length natural
sequences, from isolates, or proteolytic fragments thereof.
Further, this invention contemplates the use of isolated or
recombinant DNA, or fragments thereof, which encode proteins which
exhibit high measures of identity to an APG04, FDH02, or D1B2
protein, or which were isolated using cDNA encoding an APG04,
FDH02, or D1B2 protease protein as a probe. The isolated DNA can
have the respective regulatory sequences in the 5' and 3' flanks,
e.g., promoters, enhancers, poly-A addition signals, and
others.
[0052] IV. Making Human APG04, FDH02, or D1B2 Proteins
[0053] DNAs which encode an APG04, FDH02, or D1B2 protein or
fragments thereof can be obtained by chemical synthesis, screening
cDNA libraries, or by screening genomic libraries prepared from a
wide variety of cell lines or tissue samples.
[0054] These DNAs can be expressed in a wide variety of host cells
for the synthesis of a full-length protein or fragments which can
in turn, e.g., be used to generate polyclonal or monoclonal
antibodies; for binding studies- for construction and expression of
modified molecules; and for structure/function studies. Each of
APG04, FDH02, or D1B2, or its fragments can be expressed in host
cells that are transformed or transfected with appropriate
expression vectors. These molecules can be substantially purified
to be free of protein or cellular contaminants, other than those
derived from the recombinant host, and therefore are particularly
useful in pharmaceutical compositions when combined with a
pharmaceutically acceptable carrier and/or diluent. The antigen,
e.g., APG04, FDH02, or D1B2, or portions thereof, may be expressed
as fusions with other proteins or possessing an epitope tag.
[0055] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired antigen gene or its fragments,
usually operably linked to appropriate genetic control elements
that are recognized in a suitable host cell. The specific type of
control elements necessary to effect expression will depend upon
the eventual host cell used. Generally, the genetic control
elements can include a prokaryotic promoter system or a eukaryotic
promoter expression control system, and typically include a
transcriptional promoter, an optional operator to control the onset
of transcription, transcription enhancers to elevate the level of
mRNA expression, a sequence that encodes a suitable ribosome
binding site, and sequences that terminate transcription and
translation. Expression vectors also usually contain an-origin of
replication that allows the vector to replicate independently from
the host cell.
[0056] The vectors of this invention contain DNAs which encode an
APG04, FDH02, or D1B2 protein, or a significant fragment thereof,
typically encoding, e.g., a biologically active polypeptide, or
protein. The DNA can be under the control of a viral promoter and
can encode a selection marker. This invention further contemplates
use of such expression vectors which are capable of expressing
eukaryotic CDNA coding for an APG04, FDH02, or D1B2 protein in a
prokaryotic or eukaryotic host, where the vector is compatible with
the host and where the eukaryotic CDNA coding for the protein is
inserted into the vector such that growth of the host containing
the vector expresses the CDNA in question. Usually, expression
vectors are designed for stable replication in their host cells or
for amplification to greatly increase the total number of copies of
the desirable gene per cell. It is not always necessary to require
that an expression vector replicate in a host cell, e.g., it is
possible to effect transient expression of the protein or its
fragments in various hosts using vectors that do not contain a
replication origin that is recognized by the host cell. It is also
possible to use vectors that cause integration of an APG04, FDH02,
or D1B2 protein gene or its fragments into the host DNA by
recombination, or to integrate a promoter which controls expression
of an endogenous gene.
[0057] Vectors, as used herein, contemplate plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. Expression vectors are specialized vectors which contain
genetic control elements that effect expression of operably linked
genes. Plasmids are the most commonly used form of vector, but many
other forms of vectors which serve an equivalent function are
suitable for use herein. See, e.g., Pouwels, et al. (1985 and
Supplements) Cloning Vectors: A Laboratory Manual Elsevier, N.Y.;
and Rodriquez, et al. (eds.) (1988) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses Buttersworth, Boston, Mass.
[0058] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0059] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or its derivatives. Vectors that can be used to express
APG04, FDH02, or D1B2 proteins or fragments thereof include, but
are not limited to, such vectors as those containing the lac
promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the
pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius, et al. (1988) "Expression
Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters",
in Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses 10:205-236 Buttersworth, Boston,
Mass.
[0060] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with APG04, FDH02, or D1B2 protein sequence containing
vectors. For purposes of this invention, the most common lower
eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It
will be used generically to represent lower eukaryotes although a
number of other strains and species are also available. Yeast
vectors typically consist of a replication origin (unless of the
integrating type), a selection gene, a promoter, DNA encoding the
desired protein or its fragments, and sequences for translation
termination, polyadenylation, and transcription termination.
Suitable expression vectors for yeast include such constitutive
promoters as 3-phosphoglycerate kinase and various other glycolytic
enzyme gene promoters or such inducible promoters as the alcohol
dehydrogenase 2 promoter or metallothionine promoter. Suitable
vectors include derivatives of the following types:
self-replicating low copy number (such as the YRp-series),
self-replicating high copy number (such as the YEp-series);
integrating types (such as the YIp-series), or mini-chromosomes
(such as the YCp-series).
[0061] Higher eukaryotic tissue culture cells are typically the
preferred host cells for expression of the functionally active
APG04, FDH02, or D1B2 portease protein. In principle, many higher
eukaryotic tissue culture cell lines may be used, e.g., insect
baculovirus expression systems, whether from an invertebrate or
vertebrate source. However, mammalian cells are preferred to
achieve proper processing, both cotranslationally and
posttranslationally. Transformation or transfection and propagation
of such cells is routine. Useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell
lines, insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually include an
origin-of replication, a promoter, a translation initiation site,
RNA splice sites (e.g., if genomic DNA is used), a polyadenylation
site, and a transcription termination site. These vectors also may
contain a selection gene or amplification gene. Suitable expression
vectors may be plasmids, viruses, or retroviruses carrying
promoters derived, e.g., from such sources as from adenovirus,
SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
Representative examples of suitable expression vectors include
pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC
610.
[0062] It is likely that APG04, FDH02, or D1B2 protein need not be
glycosylated to elicit biological responses. However, it will
occasionally be desirable to express an APG04, FDH02, or D1B2
protein polypeptide in a system which provides a specific or
defined glycosylation pattern. In this case, the usual pattern will
be that provided naturally by the expression system. However, the
pattern will be modifiable by exposing the polypeptide, e.g., in
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example, an
APG04, FDH02, or D1B2 protein gene may be co-transformed with one
or more genes encoding mammalian or other glycosylating enzymes. It
is further understood that over glycosylation may be detrimental to
APG04, FDH02, or D1B2 protein biological activity, and that one of
skill may perform routine testing to optimize the degree of
glycosylation which confers optimal biological activity.
[0063] An APG04, FDH02, or D1B2 protein, or a fragment thereof, may
be engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a biologically active
form, and allows purification by standard procedures of protein
chemistry. See, e.g., Low (1989) Biochem. Biophys. Acta
988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,
et al. (1991) J. Cell Biol. 114:1275-1283.
[0064] Now that APG04, FDH02, or D1B2 proteins have been
characterized, fragments or derivatives thereof can be prepared by
conventional processes for synthesizing peptides. These include
processes such as are described in Stewart and Young (1984) Solid
Phase Peptide Synthesis Pierce Chemical Co., Rockford, Ill.;
Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis
Springer-Verlag, New York, N.Y.; and Bodanszky (1984) The
Principles of Peptide Synthesis Springer-Verlag, New York, N.Y. For
example, an azide process, an acid chloride process, an acid
anhydride process, a mixed anhydride process, an active ester
process (for example, p-nitrophenyl ester, N-hydroxysuccinimide
ester, or cyanomethyl ester), a carbodiimidazole process, an
oxidative-reductive process, or a dicyclohexylcarbodiimide
(DCCD)/additive process can be used. Solid phase and solution phase
syntheses are both applicable to the foregoing processes.
[0065] The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, for example, by extraction, precipitation,
electrophoresis and various forms of chromatography, and the like.
The APG04, FDH02, or D1B2 proteins of this invention can be
obtained in varying degrees of purity depending upon its desired
use. Purification can be accomplished by use of known protein
purification techniques or by the use of the antibodies or binding
partners herein described, e.g., in immunoabsorbant affinity
chromatography. This immunoabsorbant affinity chromatography is
carried out by first linking the antibodies to a solid support and
then contacting the linked antibodies with solubilized lysates of
appropriate source cells, lysates of other cells expressing the
ligand, or lysates or supernatants of cells producing the APG04,
FDH02, or D1B2 proteins as a result of recombinant DNA techniques,
see below.
[0066] Multiple cell lines may be screened for one which expresses
an APG04, FDH02, or D1B2 protein at a high level compared with
other cells. Various cell lines, e.g., a mouse thymic stromal cell
line TA4, is screened and selected for its favorable handling
properties. Natural APG04, FDH02, or D1B2 proteins can be isolated
from natural sources, or by expression from a transformed cell
using an appropriate expression vector. Purification of the
expressed protein is achieved by standard procedures, or may be
combined with engineered means for effective purification at high
efficiency from cell lysates or supernatants. Epitope or other
tags, e.g., FLAG or His.sub.6 segments, can be used for such
purification features.
[0067] V. Antibodies
[0068] Antibodies can be raised to various APG04, FDH02, or D1B2
proteins, including individual, polymorphic, allelic, strain, or
species variants, and fragments thereof, both in their naturally
occurring (full-length) forms and in their recombinant forms.
Additionally, antibodies can be raised to APG04, FDH02, or D1B2
proteins in either their active forms or in their inactive, e.g.,
denatured, forms. Anti-idiotypic antibodies may also be used.
[0069] A. Antibody Production
[0070] A number of immunogens may be used to produce antibodies
specifically reactive with APG04, FDH02, or D1B2 proteins.
Recombinant protein is the preferred immunogen for the production
of monoclonal or polyclonal antibodies. Naturally occurring protein
may also be used either in pure or impure form. Synthetic peptides,
made using the human APG04, FDH02, or D1B2 protein sequences
described herein, may also used as an immunogen for the production
of antibodies to APG04, FDH02, or D1B2 proteins. Recombinant
protein can be expressed in eukaryotic or prokaryotic cells as
described herein, and purified as described. Naturally folded or
denatured material can be used, as appropriate, for producing
antibodies. Either monoclonal or polyclonal antibodies may be
generated for subsequent use in immunoassays to measure the
protein.
[0071] Methods of producing polyclonal antibodies are known to
those of skill in the art. Typically, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized with the mixture. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the APG04, FDH02, or D1B2
protein of interest. When appropriately high titers of antibody to
the immunogen are obtained, usually after repeated immunizations,
blood is collected from the animal and antisera are prepared.
Further fractionation of the antisera to enrich for antibodies
reactive to the protein can be done if desired. See, e.g., Harlow
and Lane; or Coligan.
[0072] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Typically, spleen cells from
an animal immunized with a desired antigen are immortalized,
commonly by fusion with a myeloma cell (see, Kohler and Milstein
(1976) Eur. J. Immunol. 6:511-519, incorporated herein by
reference). Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according, e.g., to the
general protocol outlined by Huse, et al. (1989) Science
246:1275-1281.
[0073] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of APG04, FDH02, or D1B2
proteins can be raised by immunization of animals with conjugates
of the fragments with carrier proteins as described above.
Monoclonal antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding to normal or
defective APG04, FDH02, or D1B2 protein, or screened for agonistic
or antagonistic activity, e.g., mediated through a receptor. These
monoclonal antibodies will usually bind with at least a K.sub.D of
about 1 mM, more usually at least about 300 .mu.M, typically at
least about 10 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least
about 3 .mu.M or better.
[0074] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Harlow and Lane
(1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986)
Monoclonal Antibodies: Principles and Practice (2d ed.) Academic
Press, New York, N.Y.; and particularly in Kohler and Milstein
(1975) Nature 256:495-497, which discusses one method of generating
monoclonal antibodies. Summarized briefly, this method involves
injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0075] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse, et al.
(1989) "Generation of a Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281;
and Ward, et al. (1989) Nature 341:544-546. The polypeptides and
antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels-and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents, teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, Cabilly, U.S. Pat. No.
4,816,567; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033.
[0076] The antibodies of this invention are useful for affinity
chromatography in isolating APG04, FDH02, or D1B2 protein. Columns
can be prepared where the antibodies are linked to a solid support,
e.g., particles, such as agarose, SEPHADEX, or the like, where a
cell lysate or supernatant may be passed through the column, the
column washed, followed by increasing concentrations of a mild
denaturant, whereby purified APG04, FDH02, or D1B2 protein will be
released.
[0077] Other antibodies may block enzymatic activity. The
antibodies may also be used to screen expression libraries for
particular expression products. Usually the antibodies used in such
a procedure will be labeled with a moiety allowing easy detection
of presence of antigen by antibody binding.
[0078] Antibodies to APG04, FDH02, or D1B2 proteins may be used for
the identification of cell populations expressing APG04, FDH02, or
D1B2 protein. By assaying the expression products of cells
expressing APG04, FDH02, or D1B2 proteins it is possible to
diagnose disease, e.g., immune-compromised conditions.
[0079] Antibodies raised against each APG04, FDH02, or D1B2 protein
will also be useful to raise anti-idiotypic antibodies. These will
be useful in detecting or diagnosing various immunological
conditions related to expression of the respective antigens.
[0080] B. Immunoassays
[0081] A particular protein can be measured by a variety of
immunoassay methods. For a review of immunological and immunoassay
procedures in general, see Stites and Terr (eds.) (1991) Basic and
Clinical Immunology (7th ed.). Moreover, the immunoassays of the
present invention can be performed in many configurations, which
are reviewed extensively in Maggio (ed.) (1980) Enzyme Immunoassay
CRC Press, Boca Raton, Fla.; Tijan (1985) "Practice and Theory of
Enzyme Immunoassays," Laboratory Techniques in Biochemistry and
Molecular Biology, Elsevier Science Publishers B. V., Amsterdam;
and Harlow and Lane Antibodies. A Laboratory Manual, supra, each of
which is incorporated herein by reference. See also Chan (ed.)
(1987) Inmunoassay: A Practical Guide Academic Press, Orlando,
Fla.; Price and Newman (eds.) (1991). Principles and Practice of
Immunoassays Stockton Press, N.Y.; and Ngo (ed.) (1988)
Non-isotoric Immunoassays Plenum Press, N.Y.
[0082] Immunoassays for measurement of APG04, FDH02, or D1B2
proteins or peptides can be performed by a variety of methods known
to those skilled in the art. In brief, immunoassays to measure the
protein can be either competitive or noncompetitive binding assays.
In competitive binding assays, the sample to be analyzed competes
with a labeled analyte for specific binding sites on a capture
agent bound to a solid surface. Preferably the capture agent is an
antibody specifically reactive with APG04, FDH02, or D1B2 proteins
produced as described above. The concentration of labeled analyte
bound to the capture agent is inversely proportional to the amount
of free analyte present in the sample.
[0083] In a competitive binding immunoassay, the APG04, FDH02, or
D1B2 protein present in the sample competes with labeled protein
for binding to a specific binding agent, for example, an antibody
specifically reactive with the APG04, FDH02, or D1B2 protein. The
binding agent may be bound to a solid surface to effect separation
of bound labeled protein from the unbound labeled protein.
Alternately, the competitive binding assay may be conducted in
liquid phase and a variety of techniques known in the art may be
used to separate the bound labelled protein from the unbound
labeled protein. Following separation, the amount of bound-labeled
protein is determined. The amount of protein present in the sample
is inversely proportional to the amount of labeled protein
binding.
[0084] Alternatively, a homogeneous immunoassay may be performed in
which a separation step is not needed. In these immunoassays, the
label on the protein is altered by the binding of the protein to
its specific binding agent. This alteration in the labeled protein
results in a decrease or increase in the signal emitted by label,
so that measurement of the label at the end of the uimunoassay
allows for detection or quantitation of the protein.
[0085] APG04, FDH02, or D1B2 proteins may also be determined by a
variety of noncompetitive immunoassay methods. For example, a
two-site, solid phase sandwich immunoassay may be used. In this
type of assay, a binding agent for the protein, for example an
antibody, is attached to a solid support. A second protein binding
agent, which may also be an antibody, and which binds the protein
at a different site, is labelled. After binding at both sites on
the protein has occurred, the unbound labeled binding agent is
removed and the amount of labeled binding agent bound to the solid
phase is measured. The amount of labeled binding agent bound is
directly proportional to the amount of protein in the sample.
[0086] Western blot analysis can be used to determine the presence
of APG04, FDH02, or D1B2 proteins in a sample. Electrophoresis is
carried out, for example, on a tissue sample suspected of
containing the protein. Following electrophoresis to separate the
proteins, and transfer of the proteins to a suitable solid support,
e.g., a nitrocellulose filter, the solid support is incubated with
an antibody reactive with the protein. This antibody may be
labeled, or alternatively may be detected by subsequent incubation
with a second labeled antibody that binds the primary antibody.
[0087] The immunoassay formats described above employ labeled assay
components. The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. A wide variety of labels and methods may be used.
Traditionally, a radioactive label incorporating .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P was used.
Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation. For a review of
various labelling or signal producing systems which may be used,
see U.S. Pat. No. 4,391,904, which is incorporated herein by
reference.
[0088] Antibodies reactive with a particular protein can also be
measured by a variety of immunoassay methods. For a review of
immunological and immunoassay procedures applicable to the
measurement of antibodies by immunoassay techniques, see Stites and
Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio
(ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, A
Laboratory Manual, supra.
[0089] In brief, immunoassays to measure antisera reactive with
APG04, FDH02, or D1B2 proteins can be either competitive or
noncompetitive binding assays. In competitive binding assays, the
sample analyte competes with a labeled analyte for specific binding
sites on a capture agent bound to a solid surface. Preferably the
capture agent is a purified recombinant APG04, FDH02, or D1B2
protein produced as described above. Other sources of APG04, FDH02,
or D1B2 proteins, including isolated or partially purified
naturally occurring protein, may also be used. Noncompetitive
assays include sandwich assays, in which the sample analyte is
bound between two analyte-specific binding reagents. One of the
binding agents is used as a capture agent and is bound to a solid
surface. The second binding agent is labeled and is used to measure
or detect the resultant complex by visual or instrument means. A
number of combinations of capture agent and labelled binding agent
can be used. A variety of different immunoassay formats, separation
techniques, and labels can be also be used similar to those
described above for the measurement of APG04, FDH02, or D1B2
proteins.
[0090] VI. Purified APG04, FDH02, or D1B2 proteins
[0091] Human APG04, FDH02, or D1B2 protein amino acid sequences are
provided in SEQ ID NO: 2, 4, and 6.
[0092] Purified protein or defined peptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate polyclonal and monoclonal antibodies.
See, e.g., Coligan (1991) Current Protocols in Immunology
Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, N.Y., which are
incorporated herein by reference.
[0093] The specific binding composition can be used for screening
an expression library made from a cell line which expresses an
APG04, FDH02, or D1B2 protein. Many methods for screening are
available, e.g., standard staining of surface expressed ligand, or
by panning. Screening of intracellular expression can also be
performed by various staining or immunofluorescence procedures. The
binding compositions could be used to affinity purify or sort out
cells expressing the ligand.
[0094] The peptide segments, along with comparison to homologous
genes, can also be used to produce appropriate oligonucleotides to
screen a library. The genetic code can be used to select
appropriate oligonucleotides useful as probes for screening. In
combination with polymerase chain reaction (PCR) techniques,
synthetic oligonucleotides will-be useful in selecting desired
clones from a library, including natural allelic and polymorphic
variants.
[0095] The peptide sequences allow preparation of peptides to
generate antibodies to recognize such segments, and allow
preparation of oligonucleotides which encode such sequences. The
sequence also allows for synthetic preparation, e.g., see Dawson,
et al. (1994) Science 266:776-779. Since APG04 proteins appear to
be secreted proteins, the gene will normally possess an N-terminal
signal sequence, which is removed upon processing and secretion,
and the putative cleavage site is between amino acids 20 and 21 in
SEQ ID NO: 2 and 4, and between amino acids 19 and 20 in SEQ ID NO:
6, though it may be slightly in either direction. Analysis of the
structural features in comparison with the most closely related
reported sequences has revealed similarities with other proteins,
particularly the class of proteins known as proteases.
[0096] VII. Physical Variants
[0097] This invention also encompasses proteins or peptides having
substantial amino acid sequence similarity with an amino acid
sequence of an APG04, FDH02, or D1B2 protein. Natural variants
include individual, polymorphic, allelic, strain, or species
variants. Conservative substitutions in the amino acid sequence
will normally preserve most relevant biological activities.
[0098] Amino acid sequence similarity, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. This changes when considering
conservative substitutions as matches. Conservative substitutions
typically include substitutions within the following groups:
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Homologous amino acid
sequences include natural polymorphic, allelic, and interspecies
variations in each respective protein sequence. Typical homologous
proteins or peptides will have from 50-100% similarity (if gaps can
be introduced), to 75-100% similarity (if conservative
substitutions are included) with the amino acid sequence of the
APG04, FDH02, or D1B2 protein. Similarity measures will be at least
about 50%, generally at least about 60%, more generally at least
about 65%, usually at least about 70%, more usually at least about
75%, preferably at least about 80%, and more preferably at least
about 80%, and in particularly preferred embodiments, at least
about 85% or more. See also Needleham, et al. (1970) J. Mol. Biol.
48:443-453; Sankoff, et al. (1983) Time Warps, String Edits, and
Macromolecules: The Theory and Practice of Sequence Comrarison
Chapter One, Addison-Wesley, Reading, Mass.; and software packages
from IntelliGenetics, Mountain View, Calif.; and the University of
Wisconsin Genetics Computer Group, Madison, Wis.
[0099] Natural nucleic acids encoding mammalian APG04, FDH02, or
D1B2 proteins will typically hybridize to the nucleic acid sequence
of SEQ ID NO: 1, 3, or 5 under stringent conditions. For example,
nucleic acids encoding human APG04, FDH02, or D1B2 proteins will
normally hybridize to the nucleic acid of SEQ ID NO: 1, 3, or 5
under stringent hybridization conditions. Generally, stringent
conditions are selected to be about 10.degree. C. lower than the
thermal melting point (Tm) for the probe sequence at a defined
ionic strength and pH. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent
conditions will be those in which the salt concentration is about
0.2 molar at pH 7 and the temperature is at least about 50.degree.
C. Other factors may significantly affect the stringency of
hybridization, including, among others, base composition and size
of the complementary strands, the presence of organic solvents such
as formamide, and the extent of base mismatching. A preferred
embodiment will include nucleic acids which will bind to disclosed
sequences in 50% formamide and 200 mM NaCl at 42.degree. C.
[0100] An isolated APG04, FDH02, or D1B2 protein DNA can be readily
modified by nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and short inversions of nucleotide
stretches. These modifications result in novel DNA sequences which
encode APG04, FDH02, or D1B2 protein antigens, their derivatives,
or proteins having highly similar physiological, immunogenic, or
antigenic activity.
[0101] Modified sequences can be used to produce mutant antigens or
to enhance expression. Enhanced expression may involve gene
amplification, increased transcription, increased translation, and
other mechanisms. Such mutant APG04, FDH02, or D1B2 protein
derivatives include predetermined or site-specific mutations of the
respective protein or its fragments. "Mutant APG04, FDH02, or D1B2
protein" encompasses a polypeptide otherwise falling within the
homology definition of the human APG04, FDH02, or D1B2 protein as
set forth above, but having an amino acid sequence which differs
from that of an APG04, FDH02, or D1B2 protein as found in nature,
whether by way of deletion, substitution, or insertion. In
particular, "site specific mutant APG04, FDH02, or D1B2 protein"
generally includes proteins having significant similarity with a
protein having a sequence of SEQ ID NO: 2, 4, or 6, and as sharing
various biological activities, e.g., antigenic or immunogenic, with
those sequences, and in preferred embodiments contain most or all
of the disclosed sequence. This applies also to polymorphic
variants from different individuals. Similar concepts apply to
different APG04, FDH02, or D1B2 proteins, particularly those found
in various warm blooded animals, e.g., mammals and birds. As stated
before, it is emphasized that descriptions are generally meant to
encompass other APG04, FDH02, or D1B2 proteins, not limited to the
human embodiments specifically discussed.
[0102] Although site specific mutation sites are predetermined,
mutants need not be site specific. APG04, FDH02, or D1B2 protein
mutagenesis can be conducted by making amino acid insertions or
deletions. Substitutions, deletions, insertions, or any
combinations may be generated to arrive at a final construct.
Insertions include amino- or carboxyl-terminal fusions, e.g.
epitope tags. Random mutagenesis can be conducted at a target codon
and the expressed mutants can then be screened for the desired
activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See also, Sambrook, et al. (1989) and
Ausubel, et al. (1987 and Supplements). The mutations in the DNA
normally should not place coding sequences out of reading frames
and preferably will not create complementary regions that could
hybridize to produce secondary mRNA structure such as loops or
hairpins.
[0103] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
Thus, the fusion product of an immunoglobulin with an APG04, FDH02,
or D1B2 protein polypeptide is a continuous protein molecule having
sequences fused in a typical peptide linkage, typically made as a
single translation product and exhibiting properties derived from
each source peptide. A similar concept applies to heterologous
nucleic acid sequences.
[0104] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
protein-binding or other segments may be "swapped" between
different new fusion polypeptides or fragments. See, e.g.,
Cunningham, et al. (1989) Science 243:1330-1336; and O'Dowd, et al.
(1988) J. Biol. Chem. 263:15985-15992. Thus, new chimeric
polypeptides exhibiting new combinations of specificities will
result from the functional linkage of protein-binding specificities
and other functional domains.
[0105] VIII. Binding Agent:APG04, FDH02, or D1B2 Protein
Complexes
[0106] An APG04, FDH02, or D1B2 protein that specifically binds to
or that is specifically immunoreactive with an antibody generated
against a defined immunogen, such as an immunogen consisting of the
amino acid sequence of SEQ ID NO: 2, 4, or 6, is typically
determined in an immunoassay. The immunoassay uses a polyclonal
antiserum which was raised to a protein of SEQ ID NO: 2, 4, or 6.
This antiserum is selected to have low crossreactivity against
other proteases and any such crossreactivity is removed by
immunoabsorbtion prior to use in the immunoassay.
[0107] In order to produce antisera for use in an immunoassay, the
protein of SEQ ID NO: 2, 4, or 6, is isolated as described herein.
For example, recombinant protein may be produced in a mammalian
cell line. An inbred strain of mice such as balb/c is immunized
with the protein of SEQ ID NO: 2, 4, or 6, using a standard
adjuvant, such as Freund's adjuvant, and a standard mouse
immunization protocol (see Harlow and Lane, supra). Alternatively,
a synthetic peptide, preferably near full length, derived from the
sequences disclosed herein and conjugated to a carrier protein can
be used an immunogen. Polyclonal sera are collected and titered
against the immunogen protein in an immunoassay, for example, a
solid phase immunoassay with the immunogen immobilized on a solid
support. Polyclonal antisera with a titer of 10.sup.4 or greater
are selected and tested for their cross reactivity against other
proteases, using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573. Preferably
two proteases are used in this determination in conjunction with
either APG04, FDH02, or D1B2 protein.
[0108] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, a protein of
SEQ ID NO: 2, 4, or 6 can be immobilized to a solid support.
Proteins added to the assay compete with the binding of the
antisera to the immobilized antigen. The ability of the above
proteins to compete with the binding of the antisera to the
immobilized protein is compared to the protein of SEQ ID NO: 2, 4,
or 6. The percent crossreactivity for the above proteins is
calculated, using standard calculations. Those antisera with less
than 10% crossreactivity with each of the proteins listed above are
selected and pooled. The cross-reacting antibodies are then removed
from the pooled antisera by immunoabsorbtion with the above-listed
proteins.
[0109] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein (e.g., the protein motif of
SEQ ID NO: 2, 4, or 6). In order to make this comparison, the two
proteins are each assayed at a wide range of concentrations and the
amount of each protein required to inhibit 50% of the binding of
the antisera to the immobilized protein is determined. If the
amount of the second protein required is less than twice the amount
of the protein of SEQ ID NO: 2, 4, or 6 that is required, then the
second protein is said to specifically bind to an antibody
generated to the immunogen.
[0110] It is understood that APG04, FDH02, or D1B2 proteins are
families of homologous proteins that comprise two or more genes.
For a particular gene product, such as the human APG04, FDH02, or
D1B2 proteins, the term refers not only to the amino acid sequences
disclosed herein, but also to other proteins that are polymorphic,
allelic, non-allelic, or species variants. It is also understood
that the term "human APG04, FDH02, or D1B2 protein" includes
nonnatural mutations introduced by deliberate mutation using
conventional recombinant technology such as single site mutation,
or by excising short sections of DNA encoding APG04, FDH02, or D1B2
proteins, or by substituting new amino acids, or adding new amino
acids. Such minor alterations must substantially maintain the
immunoidentity of the original molecule and/or its biological
activity. Thus, these alterations include proteins that are
specifically immunoreactive with a designated naturally occurring
APG04, FDH02, or D1B2 protein, for example, the human APG04, FDH02,
or D1B2 protein shown in SEQ ID NO: 2, 4, or 6. The biological
properties of the altered proteins can be determined by expressing
the protein in an appropriate cell line and measuring, e.g., a
chemotactic effect. Particular protein modifications considered
minor would include conservative substitution of amino acids with
similar chemical properties, as described above for APG04, FDH02,
or D1B2 protein families as a whole. By aligning a protein
optimally with the protein of SEQ ID NO: 2, 4, or 6, and by using
the conventional immunoassays described herein to determine
immunoidentity, or by using lymphocyte chemotaxis assays, one can
determine the protein compositions of the invention.
[0111] IX. Functional Variants
[0112] The blocking of physiological response to APG04, FDH02, or
D1B2 protein may result from the inhibition of enzymatic acitvity
of the protein against its substrate, e.g., through competitive
inhibition. Thus, in vitro assays of the present invention will
often use isolated protein, membranes from cells expressing a
recombinant membrane associated proteins, soluble fragments
comprising enzymatically active segments of these proteins, or
fragments attached to solid phase substrates. These assays will
also allow for the diagnostic determination of the effects of
either binding segment mutations and modifications, or protein
mutations and modifications, e.g., protein analogs. This invention
also contemplates the use of competitive drug screening assays,
e.g., where neutralizing antibodies to antigen or receptor
fragments compete with a test compound for binding to the protein.
In this manner, the antibodies can be used to detect the presence
of a polypeptide which shares one or more antigenic binding sites
of the protein and can also be used to occupy binding sites on the
protein that might otherwise interact with a receptor.
[0113] "Derivatives" of APG04, FDH02, or D1B2 proteins include
amino acid sequence mutants, glycosylation variants, and covalent
or aggregate conjugates with other chemical moieties. Covalent
derivatives can be prepared by linkage of functionalities to groups
which are found in APG04, FDH02, or D1B2 protein amino acid side
chains or at the N- or C-termini, by means which are well known in
the art. These derivatives can include, without limitation,
aliphatic esters or amides of the carboxyl terminus, or of residues
containing carboxyl side chains, O-acyl derivatives of hydroxyl
group-containing residues, and N-acyl derivatives of the amino
terminal amino acid or amino-group containing residues, e.g.,
lysine or arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby forming
alkanoyl aroyl species. Covalent attachment to carrier proteins may
be important when immunogenic moieties are haptens.
[0114] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine, or other moieties, including
ribosyl groups or cross-linking reagents.
[0115] A major group of derivatives are covalent conjugates of the
APG04, FDH02, or D1B2 protein or fragments thereof with other
proteins or polypeptides. These derivatives can be synthesized in
recombinant culture such as N- or C-terminal fusions or by the use
of agents known in the art for their usefulness in cross-linking
proteins through reactive side groups. Preferred protein
derivatization sites with cross-linking agents are at free amino
groups, carbohydrate moieties, and cysteine residues.
[0116] Fusion polypeptides between human APG04, FDH02, or D1B2
proteins and other homologous or heterologous proteins are also
provided. Many growth factors and cytokines are homodimeric
entities, and a repeat construct may have various advantages,
including lessened susceptibility to proteolytic degradation.
Moreover, many receptors require dimerization to transduce a
signal, and various dimeric proteins or domain repeats can be
desirable. Heterologous polypeptides may be fusions between
different surface markers, resulting in, e.g., a hybrid protein
exhibiting receptor binding specificity. Likewise, heterologous
fusions may be constructed which would exhibit a combination of
properties or activities of the derivative proteins. Typical
examples are fusions of a reporter polypeptide, e.g., luciferase,
with a segment or domain of a protein, e.g., a receptor-binding
segment, so that the presence or location of the fused protein may
be easily determined. See, e.g., Dull, et al., U.S. Pat. No.
4,859,609. Other gene fusion partners include bacterial
.beta.-galactosidase, trpE, Protein A, .beta.-lactamase, alpha
amylase, alcohol dehydrogenase, and yeast alpha mating factor. See,
e.g., Godowski, et al. (1988) Science 241:812-816.
[0117] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to phosphate
groups. In some embodiments, the modifications will be useful
labeling reagents, or serve as purification targets, e.g., affinity
ligands.
[0118] This invention also contemplates the use of derivatives of
APG04, FDH02, or D1B2 proteins other than variations in amino acid
sequence or glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties. These derivatives
generally fall into the three classes: (1) salts, (2) side chain
and terminal residue covalent modifications, and (3) adsorption
complexes, for example with cell membranes. Such covalent or
aggregative derivatives are useful as immunogens, as reagents in
immunoassays, or in purification methods such as for affinity
purification of ligands or other binding ligands. For example, an
APG04, FDH02, or D1B2 protein can be immobilized by covalent
bonding to a solid support such as cyanogen bromide-activated
SEPHAROSE, by methods which are well known in the art, or adsorbed
onto polyolefin surfaces, with or without glutaraldehyde
cross-linking, for use in the assay or purification of anti-APG04,
-FDH02, or -D1B2 protein antibodies or its receptor. The APG04,
FDH02, or D1B2 proteins can also be labeled with a detectable
group, e.g., radioiodinated by the chloramine T procedure,
covalently bound to rare earth chelates, or conjugated to another
fluorescent moiety for use in diagnostic assays. Purification of
APG04, FDH02, or D1B2 proteins may be effected by immobilized
antibodies or receptor.
[0119] Isolated APG04, FDH02, or D1B2 protein genes will allow
transformation of cells lacking expression of corresponding APG04,
FDH02, or D1B2 proteins, e.g., either species types or cells which
lack corresponding proteins and exhibit negative background
activity. Expression of transformed genes will allow isolation of
antigenically pure cell lines, with defined or single specie
variants. This approach will allow for more sensitive detection and
discrimination of the physiological effects of APG04, FDH02, or
D1B2 protein substrate proteins. Subcellular fragments, e.g.,
cytoplasts or membrane fragments, can be isolated and used.
[0120] X. Uses
[0121] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for developmental abnormalities, or below
in the description of kits for diagnosis.
[0122] APG04, FDH02, or D1B2 protein nucleotides, e.g., human
APG04, FDH02, or D1B2 protein DNA or RNA, may be used as a
component in a forensic assay. For instance, the nucleotide
sequences provided may be labeled using, e.g., .sup.32P or biotin
and used to probe standard restriction fragment polymorphism blots,
providing a measurable character to aid in distinguishing between
individuals. Such probes may be used in well-known forensic
techniques such as genetic fingerprinting. In addition, nucleotide
probes made from APG04, FDH02, or D1B2 protein sequences may be
used in in situ assays to detect chromosomal abnormalities.
[0123] Antibodies and other binding agents directed towards APG04,
FDH02, or D1B2 proteins or nucleic acids may be used to purify the
corresponding APG04, FDH02, or D1B2 protein molecule. As described
in the Examples below, antibody purification of APG04, FDH02, or
D1B2 protein components is both possible and practicable.
Antibodies and other binding agents may also be used in a
diagnostic fashion to determine whether APG04, FDH02, or D1B2
protein components are present in a tissue sample or cell
population using well-known techniques described herein. The
ability to attach a binding agent to an APG04, FDH02, or D1B2
protein provides a means to diagnose disorders associated with
APG04, FDH02, or D1B2 protein misregulation. Antibodies and other
APG04, FDH02, or D1B2 protein binding agents may also be useful as
histological markers. As described in the examples below, APG04,
FDH02, or D1B2 protein expression is limited to specific tissue
types. By directing a probe, such as an antibody or nucleic acid to
an APG04, FDH02, or D1B2 protein it is possible to use the probe to
distinguish tissue and cell types in situ or in vitro.
[0124] This invention also provides reagents with significant
therapeutic value. The APG04, FDH02, or D1B2 protein (naturally
occurring or recombinant), fragments thereof, and antibodies
thereto, along with compounds identified as having binding affinity
to an APG04, FDH02, or D1B2 protein, are useful in the treatment of
conditions associated with abnormal physiology or development,
including abnormal proliferation, e.g., cancerous conditions, or
degenerative conditions. Abnormal proliferation, regeneration,
degeneration, and atrophy may be modulated by appropriate
therapeutic treatment using the compositions provided herein. The
APG04, FDH02, or D1B2 proteins likely play a role in regulation or
development of hematopoietic cells, e.g., lymphoid cells, which
affect immunological responses. Thus, for example, an antagonist of
an APG04, FDH02, or D1B2 protein could be useful in blocking the
conversion of an immature or inactive immunologically relevant
pro-protein to the mature or active form. Since these proteases
were derived from dendritic cells, antagonists could also be
important in preventing antigen processing and/or subsequent
presentation.
[0125] Other abnormal developmental conditions are known in cell
types shown to possess APG04, FDH02, or D1B2 protein mRNA by
northern blot analysis. See Berkow (ed.) The Merck Manual of
Diagnosis and Therapy, Merck & Co., Rahway, N.J.; and Thorn, et
al. Harrison's Principles of Internal Medicine. McGraw-Hill, N.Y.
Developmental or functional abnormalities, e.g., of the immune
system, cause significant medical abnormalities and conditions
which may be susceptible to prevention or treatment using
compositions provided herein.
[0126] Recombinant APG04, FDH02, or D1B2 protein antibodies can be
purified and then administered to a patient. These reagents can be
combined for therapeutic use with additional active or inert
ingredients, e.g., in conventional pharmaceutically acceptable
carriers or diluents, e.g., immunogenic adjuvants, along with
physiologically innocuous stabilizers and excipients. These
combinations can be sterile filtered and placed into dosage forms
as by lyophilization in dosage vials or storage in stabilized
aqueous preparations. This invention also contemplates use of
antibodies or binding fragments thereof, including forms which are
not complement binding.
[0127] Drug screening using antibodies or receptor or fragments
thereof can identify compounds having binding affinity to APG04,
FDH02, or D1B2 protein, including isolation of associated
components. Subsequent biological assays can then be utilized to
determine if the compound has intrinsic protease blocking activity.
Likewise, a compound having intrinsic stimulating activity might
activate the activity of an APG04, FDH02, or D1B2 protein. This
invention further contemplates the therapeutic use of antibodies to
APG04, FDH02, or D1B2 protein as antagonists. This approach should
be particularly useful with other APG04, FDH02, or D1B2 protein
polymorphic or species variants.
[0128] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman and
Gilman's: The Pharmacoloaical Bases of Therapeutics (8th ed.)
Pergamon Press; and (1990) Reminaton's Pharmaceutical Sciences
(17th ed.) Mack Publishing Co., Easton, Pa. Methods for
administration are discussed therein and below, e.g., for oral,
intravenous, intraperitoneal, or intramuscular administration,
transdermal diffusion, and others. Pharmaceutically acceptable
carriers will include water, saline, buffers, and other compounds
described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.
Dosage ranges would ordinarily be expected to be in amounts lower
than 1 mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 pM (picomolar), and most preferably less than about 1
fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous administration.
[0129] APG04, FDH02, or D1B2 proteins, fragments thereof;
antibodies to it or its fragments; antagonists; and agonists, may
be administered directly to the host to be treated or, depending on
the size of the compounds, it may be desirable to conjugate them to
carrier proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in any
conventional dosage formulation. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
See, e.g., Gilman, et al. (eds.) (1990) Goodman and Gilman's: The
Pharmacoloaical Bases of Therapeutics (8th ed.) Pergamon Press; and
(1990) Reminaton's Pharmaceutical Sciences (17th ed.) Mack
Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosaae Forms: Parenteral Medications Dekker, N.Y.;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosaae Forms:
Tablets Dekker, N.Y.; and Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y. The
therapy of this invention may be combined with or used in
association with other therapeutic agents.
[0130] Both the naturally occurring and the recombinant forms of
the APG04, FDH02, or D1B2 proteins of this invention are
particularly useful in kits and assay methods which are capable of
screening compounds for binding activity to the proteins. Several
methods of automating assays have been developed in recent years so
as to permit screening of tens of thousands of compounds in a short
period. See, e.g., Fodor, et al. (1991) Science 251:767-773, and
other descriptions of chemical diversity libraries, which describe
means for testing of binding affinity by a plurality of compounds.
The development of suitable assays can be greatly facilitated by
the availability of large amounts of purified, soluble APG04,
FDH02, or D1B2 protein as provided by this invention.
[0131] For example, antagonists can normally be found once the
protein has been structurally defined. Testing of potential protein
analogs is now possible upon the development of highly automated
assay methods using a purified receptor. In particular, new
agonists and antagonists will be discovered by using screening
techniques described herein. Of particular importance are compounds
found to have a combined blockage activity for multiple APG04,
FDH02, or D1B2 protein substrates, e.g., compounds which can serve
as antagonists for-polymorphic or species variants of an APG04,
FDH02, or D1B2 protein.
[0132] This invention is particularly useful for screening
compounds by using recombinant protein in a variety of drug
screening techniques. The advantages of using a recombinant protein
in screening for specific ligands include: (a) improved renewable
source of the APG04, FDH02, or D1B2 protein from a specific source;
(b) potentially greater number of ligands per cell giving better
signal to noise ratio in assays; and (c) species variant
specificity (theoretically giving greater biological and disease
specificity).
[0133] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing an APG04, FDH02, or D1B2
protein substrate. Cells may be isolated which express a substrate
in isolation from any others. Such cells, either in viable or fixed
form, can be used for standard enzyme/substrate cleavgage assays.
See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et
al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe
sensitive methods to detect cellular responses. Competitive assays
are particularly useful, where the cells (source of APG04, FDH02,
or D1B2 protein) are contacted and incubated with a labeled
antibody having known binding affinity to the protein, such as
.sup.125I-antibody, and a test sample whose binding affinity to the
binding composition is being measured. The bound and free labeled
binding compositions are then separated to assess the degree of
antigen binding. The amount of test compound bound is inversely
proportional to the amount of labeled receptor binding to the known
source. Any one of numerous techniques can be used to separate
bound from free antigen to assess the degree of ligand binding.
This separation step could typically involve a procedure such as
adhesion to filters followed by washing, adhesion to plastic
followed by washing, or centrifugation of the cell membranes.
Viable cells could also be used to screen for the effects of drugs
on APG04, FDH02, or D1B2 protein mediated functions, e.g.,
substrate cleavage, and others. Some detection methods allow for
elimination of a separation step, e.g., a proximity sensitive
detection system.
[0134] Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as the source of an APG04,
FDH02, or D1B2.protein. These cells are stably transformed with DNA
vectors directing the expression of an APG04, FDH02, or D1B2
protein, e.g., an engineered membrane bound form. Essentially, the
membranes would be prepared from the cells and used in a
protein/substrate cleavage assay such as the competitive assay set
forth above.
[0135] Still another approach is to use solubilized, unpurified or
solubilized, purified APG04, FDH02, or D1B2 protein from
transformed eukaryotic or prokaryotic host cells. This allows for a
"molecular" binding assay with the advantages of increased
specificity, the ability to automate, and high drug test
throughput.
[0136] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to an APG04, FDH02, or D1B2 protein,
e.g., an antibody, is described in detail in Geysen, European
Patent Application 84/03564, published on Sep. 13, 1984. First,
large numbers of different small peptide test compounds are
synthesized on a solid substrate, e.g., plastic pins or some other
appropriate surface, see Fodor, et al., supra. Then all the pins
are reacted with solubilized, unpurified or solubilized, purified
APG04, FDH02, or D1B2 protein antibody, and washed. The next step
involves detecting bound APG04, FDH02, or D1B2 protein
antibody.
[0137] Rational drug design may also be based upon structural
studies of the molecular shapes of the APG04, FDH02, or D1B2
protein and other effectors or analogs. See, e.g., Methods in
Enzymology vols 202 and 203. Effectors may be other proteins which
mediate other functions in response to antigen binding, or other
proteins which normally interact with the substrate. One means for
determining which sites interact with specific other proteins is a
physical structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crystallography Academic Press,
N.Y.
[0138] A purified APG04, FDH02, or D1B2 protein can be coated
directly onto plates for use in the aforementioned drug screening
techniques. However, non-neutralizing antibodies to these antigens
can be used as capture antibodies to immobilize the respective
antigen on the solid phase.
[0139] XI. Kits
[0140] This invention also contemplates use of APG04, FDH02, or
D1B2 proteins, fragments thereof, peptides, and their fusion
products in a variety of diagnostic kits and methods for detecting
the presence of APG04, FDH02, or D1B2 protein or an APG04, FDH02,
or D1B2 protein substrate. Typically the kit will have a
compartment containing either a defined APG04, FDH02, or D1B2
peptide or gene segment or a reagent which recognizes one or the
other, e.g., receptor fragments or antibodies.
[0141] A kit for determining the binding affinity of a test
compound to an APG04, FDH02, or D1B2 protein would typically
comprise a test compound; a labeled compound, e.g., an antibody
having known binding affinity for the APG04, FDH02, or D1B2
protein; a source of APG04, FDH02, or D1B2 protein (naturally
occurring or recombinant); and a means for separating bound from
free labeled compound, such as a solid phase for immobilizing the
APG04, FDH02, or D1B2 protein. Once compounds are screened, those
having suitable binding affinity to the APG04, FDH02, or D1B2
protein can be evaluated in suitable biological assays, as are well
known in the art, to determine whether they act as agonists or
antagonists to the substrate. The availability of recombinant
APG04, FDH02, or D1B2 polypeptides also provide well defined
standards for calibrating such assays.
[0142] A preferred kit for determining the concentration of, for
example, an APG04, FDH02, or D1B2 protein in a sample would
typically comprise a labeled compound, e.g., antibody, having known
binding affinity for the APG04, FDH02, or D1B2 protein, a source of
APG04, FDH02, or D1B2 protein (naturally occurring or recombinant),
and a means for separating the bound from free labeled compound,
for example, a solid phase for immobilizing the APG04, FDH02, or
D1B2 protein. Compartments containing reagents, and instructions,
will normally be provided.
[0143] Antibodies, including antigen binding fragments, specific
for the APG04, FDH02, or D1B2 protein, or fragments thereof, are
useful in diagnostic applications to detect the presence of
elevated levels of APG04, FDH02, or D1B2 protein and/or its
fragments. Such diagnostic assays can employ lysates, live cells,
fixed cells, immunofluorescence, cell cultures, body fluids, and
further can involve the detection of antigens related to the ligand
in serum, or the like. Diagnostic assays may be homogeneous
(without a separation step between free reagent and antigen-APG04,
-FDH02, or -D1B2 protein complex) or heterogeneous (with a
separation step). Various commercial assays exist, such as
radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA),
enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique
(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the
like. For example, unlabeled antibodies can be employed by using a
second antibody which is labeled and which recognizes the antibody
to an APG04, FDH02, or D1B2 protein or to a particular fragment
thereof. Similar assays have also been extensively discussed in the
literature. See, e.g., Harlow and Lane (1988) Antibodies: A
Laboratory Manual, CSH Press, N.Y.; Chan (ed.) (1987) Immunoassay:
A Practical Guide Academic Press, Orlando, Fla.; Price and Newman
(eds.) (1991) Principles and Practice of Immunoassay Stockton
Press, N.Y.; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum
Press, N.Y.
[0144] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against an APG04, FDH02, or D1B2 protein, as
such may be diagnostic of various abnormal states. For example,
overproduction of APG04, FDH02, or D1B2 protein may result in
production of various immunological or other medical reactions
which may be diagnostic of abnormal physiological states, e.g., in
cell growth, acitivation, or differentiation.
[0145] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody, or
labeled APG04, FDH02, or D1B2 protein is provided. This is usually
in conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after use.
Typically the kit has compartments for each useful reagent.
Desirably, the reagents are provided as a dry lyophilized powder,
where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay.
[0146] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification, or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In any of these assays, the protein, test compound, APG04, FDH02,
or D1B2 protein, or antibodies thereto can be labeled either
directly or indirectly. Possibilities for direct labeling include
label groups: radiolabels such as .sup.125I, enzymes (U.S. Pat. No.
3,645,090) such as peroxidase and alkaline phosphatase, and
fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring
the change in fluorescence intensity, wavelength shift, or
fluorescence polarization. Possibilities for indirect labeling
include biotinylation of one constituent followed by binding to
avidin coupled to one of the above label groups.
[0147] There are also numerous methods of separating the bound from
the free antigen, or alternatively the bound from the free test
compound. The APG04, FDH02, or D1B2 protein can be immobilized on
various matrices followed by washing. Suitable matrices include
plastic such as an ELISA plate, filters, and beads. Methods of
immobilizing the APG04, FDH02, or D1B2 protein to a matrix include,
without limitation, direct adhesion to plastic, use of a capture
antibody, chemical coupling, and biotin-avidin. The last step in
this approach involves the precipitation of ligand/receptor or
ligand/antibody complex by any of several methods including those
utilizing, e.g., an organic solvent such as polyethylene glycol or
a salt such as ammonium sulfate. Other suitable separation
techniques include, without limitation, the fluorescein antibody
magnetizable particle method described in Rattle, et al. (1984)
Clin. Chem. 30:1457-1461, and the double antibody magnetic particle
separation as described in U.S. Pat. No. 4,659,678.
[0148] Methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0149] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an APG04, FDH02, or D1B2 protein. These sequences can be used as
probes for detecting levels of the APG04, FDH02, or D1B2 protein
message in samples from natural sources, or patients suspected of
having an abnormal condition, e.g., immune problem. The preparation
of both RNA and DNA nucleotide sequences, the labeling of the
sequences, and the preferred size of the sequences has received
ample description and discussion in the literature. Normally an
oligonucleotide probe should have at least about 14 nucleotides,
usually at least about 18 nucleotides, and the polynucleotide
probes may be up to several kilobases. Various detectable labels
may be employed, most commonly radionuclides, particularly
.sup.32P. However, other techniques may also be employed, such as
using biotin modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionuclides, fluorophores, enzymes, or the like.
Alternatively, antibodies may be employed which can recognize
specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA
hybrid duplexes, or DNA-protein duplexes. The antibodies in turn
may be labeled and the assay carried out where the duplex is bound
to a surface, so that upon the formation of duplex on the surface,
the presence of antibody bound to the duplex can be detected. The
use of probes to the novel anti-sense RNA may be carried out using
many conventional techniques such as nucleic acid hybridization,
plus and minus screening, recombinational probing, hybrid released
translation (HRT), and hybrid arrested translation (HART). This
also includes amplification techniques such as polymerase chain
reaction (PCR).
[0150] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97.
[0151] XII. Substrate Identification
[0152] Having isolated a protease, methods exist for identifying
the target substrate. For example, a candidate substrate can be
contacted with an APG04, FDH02, or D1B2 protein in an enzymatic
reaction. The resulting cleavage product can be analyzed, e.g.,
using SDS-PAGE, HPLC, or other forms of separation. The molecular
weight of the cleavage product should be compared against the
molecular weights of the uncleaved substrate and the APG04, FDH02,
or D1B2 protein. The successful candidate substrate will exhibit a
shift to a lower molecular weight. Analysis of the substrate should
determine what site specificity may exist for the enzyme under the
tested conditions.
[0153] Alternatively, if the protease acts by transforming an
inactive substrate to the active form, the resulting activity can
be assayed, e.g., by the result of the activated factor, e.g.,
proliferation,.apoptosis, or activation of a target cell.
[0154] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the invention to specific embodiments.
EXAMPLES
[0155] I. General Methods
[0156] Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, N.Y.; Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed.) Vols. 1-3, CSH Press, N.Y.; Ausubel, et
al., Biology Greene Publishing Associates, Brooklyn, N.Y.; or
Ausubel, et al. (1987 and Supplements) Current Protocols in
Molecular Biology Wiley/Greene, N.Y.; Innis, et al. (eds.) (1990)
PCR Protocols: A Guide to Methods and Applications Academic Press,
N.Y. Methods for protein purification include such methods as
ammonium sulfate precipitation, column chromatography,
electrophoresis, centrifugation, crystallization, and others. See,
e.g., Ausubel, et al. (1987 and periodic supplements); Deutscher
(1990) "Guide to Protein Purification," Methods in Enzymology vol.
182, and other volumes in this series; and manufacturer's
literature on use of protein purification products, e.g.,
Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif.
Combination with recombinant techniques allow fusion to appropriate
segments (epitope tags), e.g., to a FLAG sequence or an equivalent
which can be fused, e.g., via a protein-removable sequence. See,
e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate Absorbent"
in Setlow (ed.) Genetic Engineering, Principle and Methods
12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) OIAexpress:
The High Level Expression & Protein Purification System
QUIAGEN, Inc., Chatsworth, Calif.
[0157] Standard immunological techniques are described, e.g., in
Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.;
and Methods in Enzymology volumes. 70, 73, 74, 84, 92, 93, 108,
116, 121, 132, 150, 162, and 163. Assays for neural cell biological
activities are described, e.g., in Wouterlood (ed. 1995)
Neuroscience Protocols modules 10, Elsevier; Methods in
Neurosciences Academic Press; and Neuromethods Humana Press,
Totowa, N.J. Methodology of developmental systems is described,
e.g., in Meisami (ed.) Handbook of Human Growth and Developmental
Biology CRC Press; and Chrispeels (ed.) Molecular Techniques and
Approaches in Developmental Biology Interscience.
[0158] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0159] II. Isolation of Human APG04, FDH02, or D1B2 Protein
[0160] A clone encoding the human APG04, FDH02, or D1B2 protein is
isolated from a natural source by many different possible methods.
Given the sequences provided herein, PCR primers or hybridization
probes are selected and/or constructed to isolate a nucleic acid,
e.g., genomic DNA segments or cDNA reverse transcripts. Appropriate
cell sources include human tissues, e.g., brain libraries. Tissue
distribution below also suggests source tissues. Genetic and
polymorphic or allelic variants are isolated by screening a
population of individuals.
[0161] PCR based detection is performed by standard methods,
preferably using approriate primers from opposite ends of the
coding sequence, but flanking segments might be selected for
specific purposes.
[0162] Alternatively, hybridization probes are selected. Particular
AT or GC contents of probes are selected depending upon the
expected homology and mismatching expected. Appropriate stringency
conditions are selected to balance an appropriate positive signal
to background ratio. Successive washing steps are used to identify
clones of greater homology.
[0163] Further clones will be isolated, e.g., using an antibody
based selection procedure. Standard expression cloning methods are
applied including, e.g., FACS staining of membrane associated
expression product. The antibodies are used to identify clones
producing a recognized protein. Alternatively, antibodies are used
to purify an APG04, FDH02, or D1B2 protein, with protein sequencing
and standard means to isolate a gene encoding that protein.
[0164] Genomic or cDNA sequence based methods will also allow for
identification of sequences naturally available, or otherwise,
which exhibit homology to the provided sequences.
[0165] III. Isolation of Mouse APG04, FDH02, or D1B2 Protein
[0166] Similar methods are used as above to isolate an appropriate
APG04, FDH02, or D1B2 protein gene. Similar source materials as
indicated above are used to isolate natural genes, including
genetic, polymorphic, allelic, or strain variants. Species variants
are also isolated using similar methods.
[0167] IV. Isolation of an Avian APG04, FDH02, or D1B2 Protein
Clone
[0168] An appropriate avian source is selected as above. Similar
methods are utilized to isolate other species variants, though the
level of similarity will typically be lower for avian APG04, FDH02,
or D1B2 protein as compared to a human to mouse sequence.
[0169] V. Expression; Purification; Characterization
[0170] With an appropriate clone from above, the coding sequence is
inserted into an appropriate expression vector. This may be in a
vector specifically selected for a prokaryote, yeast, insect, or
higher vertebrate, e.g., mammalian expression system. Standard
methods are applied to produce the gene product, preferably as a
soluble secreted molecule, but will, in certain instances, also be
made as an intracellular protein. Intracellular proteins typically
require cell lysis to recover the protein, and insoluble inclusion
bodies are a common starting material for further
purificiation.
[0171] With a clone encoding a vertebrate APG04, FDH02, or D1B2
protein, recombinant production means are used, although natural
forms may be purified from appropriate sources. The protein product
is purified by standard methods of protein purification, in certain
cases, e.g., coupled with-immunoaffinity methods. Immunoaffinity
methods are used either as a purification-step, as described above,
or as a detection assay to determine the partition properties of
the protein.
[0172] Preferably, the protein is secreted into the medium, and the
soluble product is purified from the medium in a soluble form.
Standard purification techniques applied to soluble protiens are
then applied, with enzyme assays or immunodetection methods useful
for following where the protease purifies. Alternatively, as
described above, inclusion bodies from prokaryotic expression
systems are a useful source of material. Typically, the insoluble
protein is solubilized from the inclusion bodies and refolded using
standard methods. Purification methods are developed as described
above.
[0173] In certain embodiments, the protein is made in a eukaryotic
cell which glycosylates the protein normally. The purification
methods may be affected thereby, as may biological activities. The
intact protein can be processed to release the protein domain,
probably due to a cleavage event. While recombinant protein appears
to be processed, the physiological processes which normally do such
in native cells remain to be determined.
[0174] The product of the purification method described above is
characterized to determine many structural features. Standard
physical methods are applied, e.g., amino acid analysis and protein
sequencing. The resulting protein is subjected to CD spectroscopy
and other spectroscopic methods, e.g., NMR, ESR, mass spectroscopy,
etc. The product is characterized to determine its molecular form
and size, e.g., using gel chromatography and similar techniques.
Understanding of the chromatographic properties will lead to more
gentle or efficient purification methods.
[0175] Prediction of glycosylation sites may be made, e.g., as
reported in Hansen, et al. (1995) Biochem. J. 308:801-813.
[0176] VI. Preparation of Antibodies Against Vertebrate APG04,
FDH02, or D1B2 Protein
[0177] With protein produced and purified, as above, animals are
immunized to produce antibodies. Polyclonal antiserum may be raised
using non-purified antigen, though the resulting serum will exhibit
higher background levels. Preferably, the antigen is purified using
standard protein purification techniques, including, e.g., affinity
chromatography using polyclonal serum indicated above. Presence of
specific antibodies is detected using defined synthetic peptide
fragments.
[0178] Alternatively, polyclonal serum is raised against a purified
antigen, purified as indicated above, or using synthetic peptides.
A series of overlapping synthetic peptides which encompass all of
the full length sequence, if presented to an animal, will produce
serum recognizing most linear epitopes on the protein. Such an
antiserum is used to affinity purify protein. This purified
protein, in turn, may be used to immunize another animal to produce
another antiserum preparation.
[0179] Standard techniques are used to generate induce monoclonal
antibodies to either unpurified antigen, or, preferably, purified
antigen.
[0180] VII. Cellular and Tissue Distribution
[0181] Distribution of the protein or gene products are determined,
e.g., using immunohistochemistry with an antibody reagent, as
produced above, or by screening for nucleic acids encoding the
APG04, FDH02, or D1B2 protein. Either hybridization or PCR methods
are used to detect DNA, cDNA, or message content. Histochemistry
allows determination of the specific cell types within a tissue
which express higher or lower levels of message or DNA. Antibody
techniques are useful to quantitate protein in a biological sample,
including a liquid or tissue sample. Immunoassays are developed to
quantitate protein.
[0182] Hybridization techniques were applied to the tissue types in
described above with positive or negative results, as indicated.
The commercial tissue blots may have more significant cellular
contamination from other cells, e.g., from blood or other cells
which are in the tissue.
[0183] VIII. Structure Activity Relationship
[0184] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the positions
identified above, and evaluating biological activities of the
variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0185] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analysed,
e.g., by PCR analysis and sequencing. This allows evaluation of
population polymorphisms.
[0186] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety for all purposes.
[0187] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
Sequence Submission
[0188] SEQ ID NO: 1 is human APG04 nucleotide sequence.
[0189] SEQ ID NO: 2 is human APG04 amino acid sequence.
[0190] SEQ ID NO: 3 is human FDH02 nucleotide sequence.
[0191] SEQ ID NO: 4 is human FDH02 amino acid sequence.
[0192] SEQ ID NO: 5 is human D1B2 nucleotide sequence.
[0193] SEQ ID NO: 6 is human D1B2 amino acid sequence.
Sequence CWU 1
1
7 1 2719 DNA Homo sapiens CDS (337)..(2541) 1 ttgatggcca ccaggtgatc
tctggtctct tcagtgtggc tttgcagact ataaaggcgc 60 agcgcgccaa
cgaggcgggt tggccccaga cggcggagag gaagggcaga gtcggcggtc 120
ctgagacttg gggcggcccc ttggaggtca gccccgctcg ctcctcccgg ccctctcctc
180 ctctccgagg tccgaggcgg gcagcgggct gtgggcgggc aggaggctgc
ggaggggcgg 240 ggggcaggaa ggggcggggg gctcggcgca ctcggcagga
agagaccgac ccgccacccg 300 ccgtagcccg cgcgcccctg gcactcaatc cccgcc
atg tgg ggg ctc ctg ctc 354 Met Trp Gly Leu Leu Leu 1 5 gcc ctg gcc
gcc ttc gcg ccg gcc gtc ggc ccg gct ctg ggg gcg ccc 402 Ala Leu Ala
Ala Phe Ala Pro Ala Val Gly Pro Ala Leu Gly Ala Pro 10 15 20 agg
aac tcg gtg ctg ggc ctc gcg cag ccc ggg acc acc aag gtc cca 450 Arg
Asn Ser Val Leu Gly Leu Ala Gln Pro Gly Thr Thr Lys Val Pro 25 30
35 ggc tcg acc ccg gcc ctg cat agc agc ccg gca cag ccg ccg gcg gag
498 Gly Ser Thr Pro Ala Leu His Ser Ser Pro Ala Gln Pro Pro Ala Glu
40 45 50 aca gct aac ggg acc tca gaa cag cat gtc cgg att cga gtc
atc aag 546 Thr Ala Asn Gly Thr Ser Glu Gln His Val Arg Ile Arg Val
Ile Lys 55 60 65 70 aag aaa aag gtc att atg aag aag cgg aag aag cta
act cta act cgc 594 Lys Lys Lys Val Ile Met Lys Lys Arg Lys Lys Leu
Thr Leu Thr Arg 75 80 85 ccc acc cca ctg gtg act gcc ggg ccc ctt
gtg acc ccc act cca gca 642 Pro Thr Pro Leu Val Thr Ala Gly Pro Leu
Val Thr Pro Thr Pro Ala 90 95 100 ggg acc ctc gac ccc gct gag aaa
caa gaa aca ggc tgt cct cct ttg 690 Gly Thr Leu Asp Pro Ala Glu Lys
Gln Glu Thr Gly Cys Pro Pro Leu 105 110 115 ggt ctg gag tcc ctg cga
gtt tca gat agc cgg ctt gag gca tcc agc 738 Gly Leu Glu Ser Leu Arg
Val Ser Asp Ser Arg Leu Glu Ala Ser Ser 120 125 130 agc cag tcc ttt
ggt ctt gga cca cac cga gga cgg ctc aac att cag 786 Ser Gln Ser Phe
Gly Leu Gly Pro His Arg Gly Arg Leu Asn Ile Gln 135 140 145 150 tca
ggc ctg gag gac ggc gat cta tat gat gga gcc tgg tgt gct gag 834 Ser
Gly Leu Glu Asp Gly Asp Leu Tyr Asp Gly Ala Trp Cys Ala Glu 155 160
165 gag cag gac gcc gat cca tgg ttt cag gtg gac gct ggg cac ccc acc
882 Glu Gln Asp Ala Asp Pro Trp Phe Gln Val Asp Ala Gly His Pro Thr
170 175 180 cgc ttc tcg ggt gtt atc aca cag ggc agg aac tct gtc tgg
agg tat 930 Arg Phe Ser Gly Val Ile Thr Gln Gly Arg Asn Ser Val Trp
Arg Tyr 185 190 195 gac tgg gtc aca tca tac aag gtc cag ttc agc aat
gac agt cgg acc 978 Asp Trp Val Thr Ser Tyr Lys Val Gln Phe Ser Asn
Asp Ser Arg Thr 200 205 210 tgg tgg gga agt agg aac cac agc agt ggg
atg gac gca gta ttt cct 1026 Trp Trp Gly Ser Arg Asn His Ser Ser
Gly Met Asp Ala Val Phe Pro 215 220 225 230 gcc aat tca gac cca gaa
act cca gtg ctg aac ctc ctg ccg gag ccc 1074 Ala Asn Ser Asp Pro
Glu Thr Pro Val Leu Asn Leu Leu Pro Glu Pro 235 240 245 cag gtg gcc
cgc ttc att cgc ctg ctg ccc cag acc tgg ctc cag gga 1122 Gln Val
Ala Arg Phe Ile Arg Leu Leu Pro Gln Thr Trp Leu Gln Gly 250 255 260
ggc gcg cct tgc ctc cgg gca gag atc ctg gcc tgc cca gtc tca gac
1170 Gly Ala Pro Cys Leu Arg Ala Glu Ile Leu Ala Cys Pro Val Ser
Asp 265 270 275 ccc aat gac cta ttc ctt gag gcc cct gcg tcg gga tcc
tct gac cct 1218 Pro Asn Asp Leu Phe Leu Glu Ala Pro Ala Ser Gly
Ser Ser Asp Pro 280 285 290 cta gac ttt cag cat cac aat tac aag gcc
atg agg aag ctg atg aag 1266 Leu Asp Phe Gln His His Asn Tyr Lys
Ala Met Arg Lys Leu Met Lys 295 300 305 310 cag gta caa gag caa tgc
ccc aac atc acc cgc atc tac agc att ggg 1314 Gln Val Gln Glu Gln
Cys Pro Asn Ile Thr Arg Ile Tyr Ser Ile Gly 315 320 325 aag agc tac
cag ggc ctg aag ctg tat gtg atg gaa atg tcg gac aag 1362 Lys Ser
Tyr Gln Gly Leu Lys Leu Tyr Val Met Glu Met Ser Asp Lys 330 335 340
cct ggg gag cat gag ctg ggg gag cct gag gtg cgc tac gtg gct ggc
1410 Pro Gly Glu His Glu Leu Gly Glu Pro Glu Val Arg Tyr Val Ala
Gly 345 350 355 atg cat ggg aac gag gcc ctg ggg cgg gag ttg ctt ctg
ctc ctg atg 1458 Met His Gly Asn Glu Ala Leu Gly Arg Glu Leu Leu
Leu Leu Leu Met 360 365 370 cag ttc ctg tgc cat gag ttc ctg cga ggg
aac cca cag gtg acc cgg 1506 Gln Phe Leu Cys His Glu Phe Leu Arg
Gly Asn Pro Gln Val Thr Arg 375 380 385 390 ctg ctc tct gag atg cgc
att cac ctg ctg ccc tcc atg aac cct gat 1554 Leu Leu Ser Glu Met
Arg Ile His Leu Leu Pro Ser Met Asn Pro Asp 395 400 405 ggc tat gag
atc gcc tac cac cgg ggt tca gag ctg gtg ggc tgg gcc 1602 Gly Tyr
Glu Ile Ala Tyr His Arg Gly Ser Glu Leu Val Gly Trp Ala 410 415 420
gag ggc cgc tgg aac aac cag agc atc gat ctt aac cat aat ttt gct
1650 Glu Gly Arg Trp Asn Asn Gln Ser Ile Asp Leu Asn His Asn Phe
Ala 425 430 435 gac ctc aac aca cca ctg tgg gaa gca cag gac gat ggg
aag gtg ccc 1698 Asp Leu Asn Thr Pro Leu Trp Glu Ala Gln Asp Asp
Gly Lys Val Pro 440 445 450 cac atc gtc ccc aac cat cac ctg cca ttg
ccc act tac tac acc ctg 1746 His Ile Val Pro Asn His His Leu Pro
Leu Pro Thr Tyr Tyr Thr Leu 455 460 465 470 ccc aat gcc acc gtg gct
cct gaa acg cgg gca gta atc aag tgg atg 1794 Pro Asn Ala Thr Val
Ala Pro Glu Thr Arg Ala Val Ile Lys Trp Met 475 480 485 aag cgg atc
ccc ttt gtg cta agt gcc aac ctc cac ggg ggt gag ctc 1842 Lys Arg
Ile Pro Phe Val Leu Ser Ala Asn Leu His Gly Gly Glu Leu 490 495 500
gtg gtg tcc tac cca ttc gac atg act cgc acc ccg tgg gct gcc cgc
1890 Val Val Ser Tyr Pro Phe Asp Met Thr Arg Thr Pro Trp Ala Ala
Arg 505 510 515 gag ctc acg ccc aca cca gat gat gct gtg ttt cgc tgg
ctc agc act 1938 Glu Leu Thr Pro Thr Pro Asp Asp Ala Val Phe Arg
Trp Leu Ser Thr 520 525 530 gtc tat gct ggc agt aat ctg gcc atg cag
gac acc agc cgc cga ccc 1986 Val Tyr Ala Gly Ser Asn Leu Ala Met
Gln Asp Thr Ser Arg Arg Pro 535 540 545 550 tgc cac agc cag gac ttc
tcc gtg cac ggc aac atc atc aac ggg gct 2034 Cys His Ser Gln Asp
Phe Ser Val His Gly Asn Ile Ile Asn Gly Ala 555 560 565 gac tgg cac
acg gtc ccc ggg agc atg aat gac ttc agc tac cta cac 2082 Asp Trp
His Thr Val Pro Gly Ser Met Asn Asp Phe Ser Tyr Leu His 570 575 580
acc aac tgc ttt gag gtc act gtg gag ctg tcc tgt gac aag ttc cct
2130 Thr Asn Cys Phe Glu Val Thr Val Glu Leu Ser Cys Asp Lys Phe
Pro 585 590 595 cac gag aat gaa ttg ccc cag gag tgg gag aac aac aaa
gac gcc ctc 2178 His Glu Asn Glu Leu Pro Gln Glu Trp Glu Asn Asn
Lys Asp Ala Leu 600 605 610 ctc acc tac ctg gag cag gtg cgc atg ggc
att gca gga gtg gtg agg 2226 Leu Thr Tyr Leu Glu Gln Val Arg Met
Gly Ile Ala Gly Val Val Arg 615 620 625 630 gac aag gac acg gag ctt
ggg att gct gac gct gtc att gcc gtg gat 2274 Asp Lys Asp Thr Glu
Leu Gly Ile Ala Asp Ala Val Ile Ala Val Asp 635 640 645 ggg att aac
cat gac gtg acc acg gcg tgg ggc ggg gat tat tgg cgt 2322 Gly Ile
Asn His Asp Val Thr Thr Ala Trp Gly Gly Asp Tyr Trp Arg 650 655 660
ctg ctg acc cca ggg gac tac atg gtg act gcc agt gcc gag ggc tac
2370 Leu Leu Thr Pro Gly Asp Tyr Met Val Thr Ala Ser Ala Glu Gly
Tyr 665 670 675 cat tca gtg aca cgg aac tgt cgg gtc acc ttt gaa gag
ggc ccc ttc 2418 His Ser Val Thr Arg Asn Cys Arg Val Thr Phe Glu
Glu Gly Pro Phe 680 685 690 ccc tgc aat ttc gtg ctc acc aag act ccc
aaa cag agg ctg cgc gag 2466 Pro Cys Asn Phe Val Leu Thr Lys Thr
Pro Lys Gln Arg Leu Arg Glu 695 700 705 710 ctg ctg gca gct ggg gcc
aag gtg ccc ccg gac ctt cgc agg cgc ctg 2514 Leu Leu Ala Ala Gly
Ala Lys Val Pro Pro Asp Leu Arg Arg Arg Leu 715 720 725 gag cgg cta
agg gga cag aag gat tga tacctgcggt ttaagagccc 2561 Glu Arg Leu Arg
Gly Gln Lys Asp 730 tagggcaggc tggacctgtc aagacgggaa ggggaagagt
agagagggag ggacaaagtg 2621 aggaaaaggt gctcattaaa gctaccgggc
accttaaaaa aaaaaaaaaa aaaaaaaaaa 2681 aaaaaaaaaa aaaaaaaaaa
aaaaaaaggg cggccgct 2719 2 734 PRT Homo sapiens 2 Met Trp Gly Leu
Leu Leu Ala Leu Ala Ala Phe Ala Pro Ala Val Gly 1 5 10 15 Pro Ala
Leu Gly Ala Pro Arg Asn Ser Val Leu Gly Leu Ala Gln Pro 20 25 30
Gly Thr Thr Lys Val Pro Gly Ser Thr Pro Ala Leu His Ser Ser Pro 35
40 45 Ala Gln Pro Pro Ala Glu Thr Ala Asn Gly Thr Ser Glu Gln His
Val 50 55 60 Arg Ile Arg Val Ile Lys Lys Lys Lys Val Ile Met Lys
Lys Arg Lys 65 70 75 80 Lys Leu Thr Leu Thr Arg Pro Thr Pro Leu Val
Thr Ala Gly Pro Leu 85 90 95 Val Thr Pro Thr Pro Ala Gly Thr Leu
Asp Pro Ala Glu Lys Gln Glu 100 105 110 Thr Gly Cys Pro Pro Leu Gly
Leu Glu Ser Leu Arg Val Ser Asp Ser 115 120 125 Arg Leu Glu Ala Ser
Ser Ser Gln Ser Phe Gly Leu Gly Pro His Arg 130 135 140 Gly Arg Leu
Asn Ile Gln Ser Gly Leu Glu Asp Gly Asp Leu Tyr Asp 145 150 155 160
Gly Ala Trp Cys Ala Glu Glu Gln Asp Ala Asp Pro Trp Phe Gln Val 165
170 175 Asp Ala Gly His Pro Thr Arg Phe Ser Gly Val Ile Thr Gln Gly
Arg 180 185 190 Asn Ser Val Trp Arg Tyr Asp Trp Val Thr Ser Tyr Lys
Val Gln Phe 195 200 205 Ser Asn Asp Ser Arg Thr Trp Trp Gly Ser Arg
Asn His Ser Ser Gly 210 215 220 Met Asp Ala Val Phe Pro Ala Asn Ser
Asp Pro Glu Thr Pro Val Leu 225 230 235 240 Asn Leu Leu Pro Glu Pro
Gln Val Ala Arg Phe Ile Arg Leu Leu Pro 245 250 255 Gln Thr Trp Leu
Gln Gly Gly Ala Pro Cys Leu Arg Ala Glu Ile Leu 260 265 270 Ala Cys
Pro Val Ser Asp Pro Asn Asp Leu Phe Leu Glu Ala Pro Ala 275 280 285
Ser Gly Ser Ser Asp Pro Leu Asp Phe Gln His His Asn Tyr Lys Ala 290
295 300 Met Arg Lys Leu Met Lys Gln Val Gln Glu Gln Cys Pro Asn Ile
Thr 305 310 315 320 Arg Ile Tyr Ser Ile Gly Lys Ser Tyr Gln Gly Leu
Lys Leu Tyr Val 325 330 335 Met Glu Met Ser Asp Lys Pro Gly Glu His
Glu Leu Gly Glu Pro Glu 340 345 350 Val Arg Tyr Val Ala Gly Met His
Gly Asn Glu Ala Leu Gly Arg Glu 355 360 365 Leu Leu Leu Leu Leu Met
Gln Phe Leu Cys His Glu Phe Leu Arg Gly 370 375 380 Asn Pro Gln Val
Thr Arg Leu Leu Ser Glu Met Arg Ile His Leu Leu 385 390 395 400 Pro
Ser Met Asn Pro Asp Gly Tyr Glu Ile Ala Tyr His Arg Gly Ser 405 410
415 Glu Leu Val Gly Trp Ala Glu Gly Arg Trp Asn Asn Gln Ser Ile Asp
420 425 430 Leu Asn His Asn Phe Ala Asp Leu Asn Thr Pro Leu Trp Glu
Ala Gln 435 440 445 Asp Asp Gly Lys Val Pro His Ile Val Pro Asn His
His Leu Pro Leu 450 455 460 Pro Thr Tyr Tyr Thr Leu Pro Asn Ala Thr
Val Ala Pro Glu Thr Arg 465 470 475 480 Ala Val Ile Lys Trp Met Lys
Arg Ile Pro Phe Val Leu Ser Ala Asn 485 490 495 Leu His Gly Gly Glu
Leu Val Val Ser Tyr Pro Phe Asp Met Thr Arg 500 505 510 Thr Pro Trp
Ala Ala Arg Glu Leu Thr Pro Thr Pro Asp Asp Ala Val 515 520 525 Phe
Arg Trp Leu Ser Thr Val Tyr Ala Gly Ser Asn Leu Ala Met Gln 530 535
540 Asp Thr Ser Arg Arg Pro Cys His Ser Gln Asp Phe Ser Val His Gly
545 550 555 560 Asn Ile Ile Asn Gly Ala Asp Trp His Thr Val Pro Gly
Ser Met Asn 565 570 575 Asp Phe Ser Tyr Leu His Thr Asn Cys Phe Glu
Val Thr Val Glu Leu 580 585 590 Ser Cys Asp Lys Phe Pro His Glu Asn
Glu Leu Pro Gln Glu Trp Glu 595 600 605 Asn Asn Lys Asp Ala Leu Leu
Thr Tyr Leu Glu Gln Val Arg Met Gly 610 615 620 Ile Ala Gly Val Val
Arg Asp Lys Asp Thr Glu Leu Gly Ile Ala Asp 625 630 635 640 Ala Val
Ile Ala Val Asp Gly Ile Asn His Asp Val Thr Thr Ala Trp 645 650 655
Gly Gly Asp Tyr Trp Arg Leu Leu Thr Pro Gly Asp Tyr Met Val Thr 660
665 670 Ala Ser Ala Glu Gly Tyr His Ser Val Thr Arg Asn Cys Arg Val
Thr 675 680 685 Phe Glu Glu Gly Pro Phe Pro Cys Asn Phe Val Leu Thr
Lys Thr Pro 690 695 700 Lys Gln Arg Leu Arg Glu Leu Leu Ala Ala Gly
Ala Lys Val Pro Pro 705 710 715 720 Asp Leu Arg Arg Arg Leu Glu Arg
Leu Arg Gly Gln Lys Asp 725 730 3 2030 DNA Homo sapiens CDS
(183)..(1484) 3 caggtaccgg tccggaattc ccgggtcgac ccacgcgtcc
ggtttggtgt gaggctgcga 60 gccgccgcga gttctcacgg tcccgccggc
gccaccaccg cggtcactca ccgccgccgc 120 cgccaccact gccaccacgg
tcgcctgcca caggtgtctg caattgaact ccaaggtgca 180 ga atg gtt tgg aaa
gta gct gta ttc ctc agt gtg gcc ctg ggc att 227 Met Val Trp Lys Val
Ala Val Phe Leu Ser Val Ala Leu Gly Ile 1 5 10 15 ggt gcc gtt cct
ata gat gat cct gaa gat gga ggc aag cac tgg gtg 275 Gly Ala Val Pro
Ile Asp Asp Pro Glu Asp Gly Gly Lys His Trp Val 20 25 30 gtg atc
gtg gca ggt tca aat ggc tgg tat aat tat agg cac cag gca 323 Val Ile
Val Ala Gly Ser Asn Gly Trp Tyr Asn Tyr Arg His Gln Ala 35 40 45
gac gcg tgc cat gcc tac cag atc att cac cgc aat ggg att cct gac 371
Asp Ala Cys His Ala Tyr Gln Ile Ile His Arg Asn Gly Ile Pro Asp 50
55 60 gaa cag atc gtt gtg atg atg tac gat gac att gct tac tct gaa
gac 419 Glu Gln Ile Val Val Met Met Tyr Asp Asp Ile Ala Tyr Ser Glu
Asp 65 70 75 aat ccc act cca gga att gtg atc aac agg ccc aat ggc
aca gat gtc 467 Asn Pro Thr Pro Gly Ile Val Ile Asn Arg Pro Asn Gly
Thr Asp Val 80 85 90 95 tat cag gga gtc ccg aag gac tac act gga gag
gat gtt acc cca caa 515 Tyr Gln Gly Val Pro Lys Asp Tyr Thr Gly Glu
Asp Val Thr Pro Gln 100 105 110 aat ttc ctt gct gtg ttg aga ggc gat
gca gaa gca gtg aag ggc ata 563 Asn Phe Leu Ala Val Leu Arg Gly Asp
Ala Glu Ala Val Lys Gly Ile 115 120 125 gga tcc ggc aaa gtc ctg aag
agt ggc ccc cag gat cac gtg ttc att 611 Gly Ser Gly Lys Val Leu Lys
Ser Gly Pro Gln Asp His Val Phe Ile 130 135 140 tac ttc act gac cat
gga tct act gga ata ctg gtt ttt ccc aat gaa 659 Tyr Phe Thr Asp His
Gly Ser Thr Gly Ile Leu Val Phe Pro Asn Glu 145 150 155 gat ctt cat
gta aag gac ctg aat gag acc atc cat tac atg tac aaa 707 Asp Leu His
Val Lys Asp Leu Asn Glu Thr Ile His Tyr Met Tyr Lys 160 165 170 175
cac aaa atg tac cga aag atg gtg ttc tac att gaa gcc tgt gag tct 755
His Lys Met Tyr Arg Lys Met Val Phe Tyr Ile Glu Ala Cys Glu Ser 180
185 190 ggg tcc atg atg aac cac ctg ccg gat aac atc aat gtt tat gca
act 803 Gly Ser Met Met Asn His Leu Pro Asp Asn Ile Asn Val Tyr Ala
Thr 195 200 205 act gct gcc aac ccc aga gag tcg tcc tac gcc tgt tac
tat gat gag 851 Thr Ala Ala Asn Pro Arg Glu Ser Ser Tyr Ala Cys Tyr
Tyr Asp Glu 210 215 220 aag agg tcc acg tac ctg ggg gac tgg tac agc
gtc aac tgg atg gaa 899 Lys Arg Ser Thr Tyr Leu Gly Asp Trp Tyr Ser
Val Asn Trp Met Glu 225 230 235 gac tcg gac gtg gaa gat ctg act
aaa
gag acc ctg cac aag cag tac 947 Asp Ser Asp Val Glu Asp Leu Thr Lys
Glu Thr Leu His Lys Gln Tyr 240 245 250 255 cac ctg gta aaa tcg cac
acc aac acc agc cac gtc atg cag tat gga 995 His Leu Val Lys Ser His
Thr Asn Thr Ser His Val Met Gln Tyr Gly 260 265 270 aac aaa aca atc
tcc acc atg aaa gtg atg cag ttt cag ggt atg aaa 1043 Asn Lys Thr
Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly Met Lys 275 280 285 cgc
aaa gcc agt tct ccc gtc ccc cta cct cca gtc aca cac ctt gac 1091
Arg Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr His Leu Asp 290
295 300 ctc acc ccc agc cct gat gtg cct ctc acc atc atg aaa agg aaa
ctg 1139 Leu Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met Lys Arg
Lys Leu 305 310 315 atg aac acc aat gat ctg gag gag tcc agg cag ctc
acg gag gag atc 1187 Met Asn Thr Asn Asp Leu Glu Glu Ser Arg Gln
Leu Thr Glu Glu Ile 320 325 330 335 cag cgg cat ctg gat gcc agg cac
ctc att gag aag tca gtg cgt aag 1235 Gln Arg His Leu Asp Ala Arg
His Leu Ile Glu Lys Ser Val Arg Lys 340 345 350 atc gtc tcc ttg ctg
gca gcg tcc gag gct gag gtg gag cag ctc ctg 1283 Ile Val Ser Leu
Leu Ala Ala Ser Glu Ala Glu Val Glu Gln Leu Leu 355 360 365 tcc gag
aga gcc ccg ctc acg ggg cac agc tgc tac cca gag gcc ctg 1331 Ser
Glu Arg Ala Pro Leu Thr Gly His Ser Cys Tyr Pro Glu Ala Leu 370 375
380 ctg cac ttc cgg acc cac tgc ttc aac tgg cac tcc ccc acg tac gag
1379 Leu His Phe Arg Thr His Cys Phe Asn Trp His Ser Pro Thr Tyr
Glu 385 390 395 tat gcg ttg aga cat ttg tac gtg ctg gtc aac ctt tgt
gag aag ccg 1427 Tyr Ala Leu Arg His Leu Tyr Val Leu Val Asn Leu
Cys Glu Lys Pro 400 405 410 415 tat cca ctt cac agg ata aaa ttg tcc
atg gac cac gtg tgc ctt ggt 1475 Tyr Pro Leu His Arg Ile Lys Leu
Ser Met Asp His Val Cys Leu Gly 420 425 430 cac tac tga agagctgcct
cctggaagct tttccaagtg tgagcgcccc 1524 His Tyr cccgactgtg tgctgatcag
agactggaga ggtggagtga gaagtctccg ctgctcgggc 1584 cctcctgggg
agcccccgct ccagggctcg ctccaggacc ttcttcacaa gatgacttgc 1644
tcgctgttac ctgcttcccc agtcttttct gaaaaactac aaattagggt gggaaaagct
1704 ctgtattgag aagggtcata tttgctttct aggaggtttg ttgttttgcc
tgttagtttt 1764 gaggagcagg aagctcatgg gggcttctgt agcccctctc
aaaaggagtc tttattctga 1824 gaatttgaag ctgaaacctc tttaaatttt
cagaatgatt ttattgaaga gggccgcaag 1884 ccccaaatgg aaaactgttt
ttagaaaata tgatgatttt tgattgcttt tgtatttaat 1944 tctgcaggtg
ttcaagtctt aaaaaataaa gatttataac agaacccaaa aaaaaaaaaa 2004
aaaaaaaaaa aaaaaagggc ggccgc 2030 4 433 PRT Homo sapiens 4 Met Val
Trp Lys Val Ala Val Phe Leu Ser Val Ala Leu Gly Ile Gly 1 5 10 15
Ala Val Pro Ile Asp Asp Pro Glu Asp Gly Gly Lys His Trp Val Val 20
25 30 Ile Val Ala Gly Ser Asn Gly Trp Tyr Asn Tyr Arg His Gln Ala
Asp 35 40 45 Ala Cys His Ala Tyr Gln Ile Ile His Arg Asn Gly Ile
Pro Asp Glu 50 55 60 Gln Ile Val Val Met Met Tyr Asp Asp Ile Ala
Tyr Ser Glu Asp Asn 65 70 75 80 Pro Thr Pro Gly Ile Val Ile Asn Arg
Pro Asn Gly Thr Asp Val Tyr 85 90 95 Gln Gly Val Pro Lys Asp Tyr
Thr Gly Glu Asp Val Thr Pro Gln Asn 100 105 110 Phe Leu Ala Val Leu
Arg Gly Asp Ala Glu Ala Val Lys Gly Ile Gly 115 120 125 Ser Gly Lys
Val Leu Lys Ser Gly Pro Gln Asp His Val Phe Ile Tyr 130 135 140 Phe
Thr Asp His Gly Ser Thr Gly Ile Leu Val Phe Pro Asn Glu Asp 145 150
155 160 Leu His Val Lys Asp Leu Asn Glu Thr Ile His Tyr Met Tyr Lys
His 165 170 175 Lys Met Tyr Arg Lys Met Val Phe Tyr Ile Glu Ala Cys
Glu Ser Gly 180 185 190 Ser Met Met Asn His Leu Pro Asp Asn Ile Asn
Val Tyr Ala Thr Thr 195 200 205 Ala Ala Asn Pro Arg Glu Ser Ser Tyr
Ala Cys Tyr Tyr Asp Glu Lys 210 215 220 Arg Ser Thr Tyr Leu Gly Asp
Trp Tyr Ser Val Asn Trp Met Glu Asp 225 230 235 240 Ser Asp Val Glu
Asp Leu Thr Lys Glu Thr Leu His Lys Gln Tyr His 245 250 255 Leu Val
Lys Ser His Thr Asn Thr Ser His Val Met Gln Tyr Gly Asn 260 265 270
Lys Thr Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly Met Lys Arg 275
280 285 Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr His Leu Asp
Leu 290 295 300 Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met Lys Arg
Lys Leu Met 305 310 315 320 Asn Thr Asn Asp Leu Glu Glu Ser Arg Gln
Leu Thr Glu Glu Ile Gln 325 330 335 Arg His Leu Asp Ala Arg His Leu
Ile Glu Lys Ser Val Arg Lys Ile 340 345 350 Val Ser Leu Leu Ala Ala
Ser Glu Ala Glu Val Glu Gln Leu Leu Ser 355 360 365 Glu Arg Ala Pro
Leu Thr Gly His Ser Cys Tyr Pro Glu Ala Leu Leu 370 375 380 His Phe
Arg Thr His Cys Phe Asn Trp His Ser Pro Thr Tyr Glu Tyr 385 390 395
400 Ala Leu Arg His Leu Tyr Val Leu Val Asn Leu Cys Glu Lys Pro Tyr
405 410 415 Pro Leu His Arg Ile Lys Leu Ser Met Asp His Val Cys Leu
Gly His 420 425 430 Tyr 5 1173 DNA Homo sapiens CDS (1)..(1173) 5
atg cgc ggt ctc ggg ctc tgg ctg ctg ggc gcg atg atg ctg cct gcg 48
Met Arg Gly Leu Gly Leu Trp Leu Leu Gly Ala Met Met Leu Pro Ala 1 5
10 15 att gcc ccc agc cgg ccc tgg gcc ctc atg gag cag tat gag gtc
gtg 96 Ile Ala Pro Ser Arg Pro Trp Ala Leu Met Glu Gln Tyr Glu Val
Val 20 25 30 ttg ccg cgg tgt ctg cca ggc ccc cga gtc cgc cga gct
ctg ccc tcc 144 Leu Pro Arg Cys Leu Pro Gly Pro Arg Val Arg Arg Ala
Leu Pro Ser 35 40 45 cac ttg ggc ctg cac cca gag agg gtg agc tac
gtc ctt ggg gcc aca 192 His Leu Gly Leu His Pro Glu Arg Val Ser Tyr
Val Leu Gly Ala Thr 50 55 60 ggg cac aac ttc acc ctc cac ctg cgg
aag aac agg gac ctg ctg ggt 240 Gly His Asn Phe Thr Leu His Leu Arg
Lys Asn Arg Asp Leu Leu Gly 65 70 75 80 tcc ggc tac aca gag acc tat
acg gct gcc aat ggc tcc gag gtg acg 288 Ser Gly Tyr Thr Glu Thr Tyr
Thr Ala Ala Asn Gly Ser Glu Val Thr 85 90 95 gag cag cct cgc ggg
cag gac cac tgc ttc tac cag ggc cac gta gag 336 Glu Gln Pro Arg Gly
Gln Asp His Cys Phe Tyr Gln Gly His Val Glu 100 105 110 ggg tac ccg
gac tca gcc gcc agc ctc agc acc tgt gcc ggc ctc agg 384 Gly Tyr Pro
Asp Ser Ala Ala Ser Leu Ser Thr Cys Ala Gly Leu Arg 115 120 125 ggt
ttc ttc cag gtg ggg tca gac ctg cac ctg atc gag ccc ctg gat 432 Gly
Phe Phe Gln Val Gly Ser Asp Leu His Leu Ile Glu Pro Leu Asp 130 135
140 gaa ggt ggc gag ggc gga cgg cac gcc gtg tac cag gct gag cac ctg
480 Glu Gly Gly Glu Gly Gly Arg His Ala Val Tyr Gln Ala Glu His Leu
145 150 155 160 ctg cag acg gcc ggg acc tgc ggg gtc agc gac gac agc
ctg ggc agc 528 Leu Gln Thr Ala Gly Thr Cys Gly Val Ser Asp Asp Ser
Leu Gly Ser 165 170 175 ctc ctg gga ccc cgg acg gca gcc gtc ttc agg
cct cgg ccc ggg gac 576 Leu Leu Gly Pro Arg Thr Ala Ala Val Phe Arg
Pro Arg Pro Gly Asp 180 185 190 tct ctg cca tcc cga gag acc cgc tac
gtg gag ctg tat gtg gtc gtg 624 Ser Leu Pro Ser Arg Glu Thr Arg Tyr
Val Glu Leu Tyr Val Val Val 195 200 205 gac aat gca gag ttc cag atg
ctg ggg agc gaa gca gcc gtg cgt cat 672 Asp Asn Ala Glu Phe Gln Met
Leu Gly Ser Glu Ala Ala Val Arg His 210 215 220 cgg gtg ctg gag gtg
gtg aat cac gtg gac aag cta tat cag aaa ctc 720 Arg Val Leu Glu Val
Val Asn His Val Asp Lys Leu Tyr Gln Lys Leu 225 230 235 240 aac ttc
cgt gtg gtc ctg gtg ggc ctg gag att tgg aat agt cag gac 768 Asn Phe
Arg Val Val Leu Val Gly Leu Glu Ile Trp Asn Ser Gln Asp 245 250 255
agg ttc cac gtc agc ccc gac ccc agt gtc aca ctg gag aac ctc ctg 816
Arg Phe His Val Ser Pro Asp Pro Ser Val Thr Leu Glu Asn Leu Leu 260
265 270 acc tgg cag gca cgg caa cgg aca cgg cgg cac ctg cat gac aac
gta 864 Thr Trp Gln Ala Arg Gln Arg Thr Arg Arg His Leu His Asp Asn
Val 275 280 285 cag ctc atc acg ggt gtc gac ttc acc ggg act act gtg
ggg ttt gcc 912 Gln Leu Ile Thr Gly Val Asp Phe Thr Gly Thr Thr Val
Gly Phe Ala 290 295 300 agg gtg tcc gcc atg tgc tcc cac agc tca ggg
gct gtg aac cag gac 960 Arg Val Ser Ala Met Cys Ser His Ser Ser Gly
Ala Val Asn Gln Asp 305 310 315 320 cac agc aag aac ccc gtg ggc gtg
gct gca cca tgg ccc atg aga tgg 1008 His Ser Lys Asn Pro Val Gly
Val Ala Ala Pro Trp Pro Met Arg Trp 325 330 335 gcc aca acc tgg gca
tgg acc atg atg aga acg tcc agg gct gcc gct 1056 Ala Thr Thr Trp
Ala Trp Thr Met Met Arg Thr Ser Arg Ala Ala Ala 340 345 350 gcc agg
aac gct tcg agg ccg gcc gct gca tca tgg cag gca gca ttg 1104 Ala
Arg Asn Ala Ser Arg Pro Ala Ala Ala Ser Trp Gln Ala Ala Leu 355 360
365 gct cca gtt tcc cca gga tgt tca gtg act gca gcc agg cct acc tgg
1152 Ala Pro Val Ser Pro Gly Cys Ser Val Thr Ala Ala Arg Pro Thr
Trp 370 375 380 aga gct ttt tgg agc ggc cgc 1173 Arg Ala Phe Trp
Ser Gly Arg 385 390 6 391 PRT Homo sapiens 6 Met Arg Gly Leu Gly
Leu Trp Leu Leu Gly Ala Met Met Leu Pro Ala 1 5 10 15 Ile Ala Pro
Ser Arg Pro Trp Ala Leu Met Glu Gln Tyr Glu Val Val 20 25 30 Leu
Pro Arg Cys Leu Pro Gly Pro Arg Val Arg Arg Ala Leu Pro Ser 35 40
45 His Leu Gly Leu His Pro Glu Arg Val Ser Tyr Val Leu Gly Ala Thr
50 55 60 Gly His Asn Phe Thr Leu His Leu Arg Lys Asn Arg Asp Leu
Leu Gly 65 70 75 80 Ser Gly Tyr Thr Glu Thr Tyr Thr Ala Ala Asn Gly
Ser Glu Val Thr 85 90 95 Glu Gln Pro Arg Gly Gln Asp His Cys Phe
Tyr Gln Gly His Val Glu 100 105 110 Gly Tyr Pro Asp Ser Ala Ala Ser
Leu Ser Thr Cys Ala Gly Leu Arg 115 120 125 Gly Phe Phe Gln Val Gly
Ser Asp Leu His Leu Ile Glu Pro Leu Asp 130 135 140 Glu Gly Gly Glu
Gly Gly Arg His Ala Val Tyr Gln Ala Glu His Leu 145 150 155 160 Leu
Gln Thr Ala Gly Thr Cys Gly Val Ser Asp Asp Ser Leu Gly Ser 165 170
175 Leu Leu Gly Pro Arg Thr Ala Ala Val Phe Arg Pro Arg Pro Gly Asp
180 185 190 Ser Leu Pro Ser Arg Glu Thr Arg Tyr Val Glu Leu Tyr Val
Val Val 195 200 205 Asp Asn Ala Glu Phe Gln Met Leu Gly Ser Glu Ala
Ala Val Arg His 210 215 220 Arg Val Leu Glu Val Val Asn His Val Asp
Lys Leu Tyr Gln Lys Leu 225 230 235 240 Asn Phe Arg Val Val Leu Val
Gly Leu Glu Ile Trp Asn Ser Gln Asp 245 250 255 Arg Phe His Val Ser
Pro Asp Pro Ser Val Thr Leu Glu Asn Leu Leu 260 265 270 Thr Trp Gln
Ala Arg Gln Arg Thr Arg Arg His Leu His Asp Asn Val 275 280 285 Gln
Leu Ile Thr Gly Val Asp Phe Thr Gly Thr Thr Val Gly Phe Ala 290 295
300 Arg Val Ser Ala Met Cys Ser His Ser Ser Gly Ala Val Asn Gln Asp
305 310 315 320 His Ser Lys Asn Pro Val Gly Val Ala Ala Pro Trp Pro
Met Arg Trp 325 330 335 Ala Thr Thr Trp Ala Trp Thr Met Met Arg Thr
Ser Arg Ala Ala Ala 340 345 350 Ala Arg Asn Ala Ser Arg Pro Ala Ala
Ala Ser Trp Gln Ala Ala Leu 355 360 365 Ala Pro Val Ser Pro Gly Cys
Ser Val Thr Ala Ala Arg Pro Thr Trp 370 375 380 Arg Ala Phe Trp Ser
Gly Arg 385 390 7 8 PRT Artificial Sequence The source of this
sequence is Scott, et al. (1992) Proc. Natl. A cad. Sci. USA
89658-662, which is cited in Abe, et. al., on page 15, lines 29-30,
of the Specification. The sequence is derived from "G4 glycinin." 7
Glu Thr Arg Asn Gly Val Glu Glu 1 5
* * * * *