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1. WO1996038725 - ATTACHMENT ENHANCED 293 CELLS

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ATTACHMENT ENHANCED 293 CELLS

Field of the Invention
This invention relates generally to cell lines used in the recombinant production, screening or measurement of protein or protein interactions in vitro.

Background of the Invention
The primary human embryonic kidney (HEK) 293 cell line is a permanent line of cells transformed by sheared human adenovirus type 5 (Ad 5) DNA. The cells are particularly sensitive to human adenovirus, are highly permissive for adenovirus DNA, and contain and express the transforming genes of Ad5. This is a hypotriploid human cell line. See, F. Graham et al., J. Gen. Virol.. 36:59-72 (1977);

T. Harrison et al., Virology. 77:319-329 (1977).
This cell line, which is readily available from commercial sources, such as the American Type Culture Collection, is used extensively in in vitro assays, and for the production of recombinant proteins and viruses. However, in washing steps which are conventionally and repeatedly employed in such in vitro assays and other manipulations of these cells, the cells readily detach or are washed away from the plates or dishes in which the studies are performed. This problem typically results in inaccurate, unreliably low measurement or collection of the protein, peptide or interaction to which the assay is directed.
There remains a need in the art for a cell substrate useful in in vitro manipulations in genetic engineering, which permits the measurement of accurate results.

Brief Summary of the Invention
In one aspect, the invention provides improved HEK 293 cells, which cells are 293 cells which have been transfected with a mammalian macrophage scavenger receptor gene. Preferably, this gene is the human Type I or II macrophage scavenger receptor gene [SEQ ID NOS: 1 or 3].
In another aspect, the invention provides a method of enhancing the ability of

HEK 293 cells to attach in tissue culture. This method involves the steps of transfecting 293 cells with a selected mammalian macrophage scavenger receptor gene.
In yet another aspect, the invention provides a method of screening compounds for biological activity which involves screening the improved 293 cells of the invention. In this method, the improved 293 cells have been further transfected with a selected gene and are then screened for expression of the selected gene. The cells expressing the selected genes are incubated in the presence of a compound of unknown biological activity, and then screened for the ability of the compound to affect the expressed gene product or its function.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings
Fig. 1 provides the nucleic acid [SEQ ID NO:l] and amino acid [SEQ ID NO:2] sequences of the human macrophage scavenger receptor type I.
Fig. 2 provides the nucleic acid [SEQ ID NO:3] and amino acid [SEQ ID NO:4] sequences of the human macrophage scavenger receptor type II.

Detailed Description of the Invention
The present invention provides an improved human embryonic kidney cell line, 293. The inventors have surprisingly found that human embryonic kidney (HEK) 293 cells transfected with a mammalian macrophage scavenger receptor gene demonstrate an enhanced ability to attach to a solid support as compared to conventional, unmodified 293 cells. In contrast to unmodified 293 cells, the improved 293 cells of the invention are not as readily washed away as unmodified 293 cells under the normal conditions of biological assays. Thus, the improved 293 cells of the invention are particularly well suited for use in in vitro studies and other applications for which unmodified 293 cells may be used.
As used herein "solid support" is any surface used for culturing, for in vitro assays, and the like. For example, a typical solid support is a plastic tissue culture plate, or a multi-well plate, hollow fibers, a test tube, conventionally employed plastic beads, glass beads, etc. Other solid supports are well known to those of skill in the art.
By "enhanced ability to attach" is meant that the transfected cells of this invention attach to the solid support with sufficient avidity to resist detachment which normally occurs with untransfected 293 cells caused by assay washing steps with buffer or growth medium. More specifically, the transfected cells of this invention because of the characteristic of enhanced attachment provide results of, for example, five times the cell number remaining after two washes as compared to the number of cells remaining following two washes of untransfected cells.
The human embryonic kidney cell line, 293, is readily available from the

American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S.A., under accession number ATCC CRL 1573. Also encompassed by this invention are progeny and derivatives of this cell line, which may be prepared using conventional techniques. See, Sambrook, Molecular Cloning: A Laboratory
Manual. 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).
According to this invention, these cells are modified by transfection with a selected mammalian macrophage scavenger receptor (MSR) gene. Currently, in a preferred embodiment, this gene is selected from a human MSR Type I or Type II gene, and most preferably, the gene is characterized by the sequence provided in GenBank, under accession number D90187 (MSR Type I) or D90188 (MSR Type II). The sequences [SEQ ID NO:l and 2] of MSR Type I are provided in Fig. 1. The sequences [SEQ ID NO: 3 and 4] of MSR Type II are provided in Fig. 2. Both of these genes were obtained from the human monocytic cell line THR-1 following four days of phorbol ester treatment. These two gene sequences are differential splice variants of a single human gene, and are described in more detail in A.
Matsumoto et al, Proc. Natl. Acad. Sci. USA. 87:9133-9137 (1990), incorporated by reference herein.
It is anticipated that non-human homologs of MSR I or MSR II will be similarly useful in preparing the improved 293 cells according to the invention. Particularly desirable are the bovine [T. Kodama et al, Proc. Natl. Acad. Sci. USA. 85:9238-9242 (1988)], murine [M. Freeman et al, Proc. Natl. Acad. Sci. USA.

87:8810-8814 (1990)] and rabbit [P. E. Bickel and M. W. Freeman, J. Clin. Invest.. 90: 1450-1457 (1992)] homologs, each of which is at least 60-80% homologous with the human MSR genes. It is further anticipated that other human scavenger receptor genes, particularly other genes which are produced recombinantly or are
differentially selective for oxidized or acetylation-modified low density lipoprotein (LDL) species or another desired lipoprotein species, will be similarly useful.
One of these genes, preferably a human MSR gene, is selected and cloned into an appropriate vector for use in transfecting the 293 cells. Generally, a suitable expression vector is one which contains control or regulatory sequences operably linked with the nucleic acid sequences of the gene. These regulatory sequences are capable of directing the expression of the gene product in the 293 cells. Suitable vectors and regulatory sequences are well known to those of skill in the art and this invention is not limited by the selection thereof.
For example, suitable vectors may be, or contain components from, viral vectors selected from simian virus SN40, retroviruses, bovine papilloma virus, vaccinia virus, and adenovirus, or commonly used bacterial vectors or commonly used mammalian expression vectors or integrative vectors which lead to a stable expression cell line. The vector used in the examples below is pCDΝ [Ν. Aiyar et al, Mol. Cell. Biochem.. 131:75-96 (1994)], which contains the promoter from cytomegalovirus, followed by a polycloning site and a polyadenylation site, the SN40 early enhancer, the human gene for dihydrofolate reductase, and a gene conferring resistance to neomycin.
Methods for introduction of a vector containing an MSR gene into mammalian cells are well known. Examples of suitable methods include, without limitation, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DΝA into nuclei.
Sequences which contain selectable markers may also be transfected into the cell line. These markers may be contained on the vector containing the MSR gene, or may be separately transfected using conventional techniques, such as those described herein. Selectable markers for mammalian cells are known in the art, and include for example, thymidine kinase, dihydrofolate reductase (together with methotrexate as a DHFR amplifier), aminoglycoside phosphotransferase, hydromycin B phosphotransferase, asparagine synthetase, adenosine deaminase, metallothionien, and antibiotic resistant genes such as neomycin. Other markers may be readily selected by one of skill in the art, as desired.
As described in more detail below, if the MSR transfected cell is desired for use in a screening assay, the cell may also be transfected with other genes. The additional gene(s) may, for example, encode a protein which will be screened for biological activity or for interaction with the MSR or another transfected gene.
Following transfection with the selected MSR gene (and optionally, any other gene), the cells are incubated in a suitable selection medium, e.g., Eagles MEM, Dulbecco's MEM or the like.
Once modified to contain the MSR gene, or another suitable gene, according to the methods described above, the improved 293 cells are particularly well suited for use in any assay in which an unmodified 293 cell may be used. However the use of the improved 293 cells of the invention will result in superior attachment, and thus, more accurate test results.
An exemplary use of the improved 293 cells of the invention includes the use of these cells in a method of screening compounds for biological activity. This method involves the use of the attachment enhanced 293 cells of the invention which have been further transfected with a selected gene sequence. These cells are subsequently screened for expression of the selected gene. The cells expressing these selected genes are then incubated in the presence of a compound of unknown biological activity and further assayed for the ability of the compound to affect the expressed gene product.
Similarly, the attachment enhanced 293 cells of the invention may be used to identify antagonists of the MSR gene, i.e., to develop agents for atherosclerosis. Suitable assays for identifying antagonists to an expressed gene product are well known to those of skill in the art. See, T. Kodama et al, Nature.243:531-535
(1990), A.M. Pearson et al, J. Biol. Chem.. 268:3554 (1993).

The surprising result of enhanced attachment demonstrated by 293 cells transfected with MSR genes is not demonstrated when other cells, such as Chinese Hamster Ovary (CHO) cells, are transfected with MSR I or MSR II. To the inventors' knowledge, no other cell line has demonstrated this result when transfected with MSR genes.
The following examples illustrate the preferred methods for preparing the modified 293 cells of the invention and uses therefor. These examples are illustrative only and are not intended to limit the scope of the invention.

Example 1 - Calcium phosphate transfection of macrophage scavenger receptor I and II into human embryonic kidney 293 cells
The macrophage scavenger receptor I or II cDNAs [SEQ ID NO: 1 and 3, respectively] were subcloned into the mammalian expression vector pCDN in the correct orientation [N. Aiyar, Mol. Cell. Biochem..131:75-86 (1994)].
The resulting construct containing the macrophage scavenger receptor I or II cDNA was used to transfect human embryonic kidney (HEK) 293 cells by calcium phosphate transfection. One day prior to the transfection, the HEK 293 cells were plated into 10 cm dishes at a density of 2 x 105 cells, so that the cells would be approximately 10% confluent within 24 hours. The cells were seeded into Eagle's Minimal Essential Medium (EMEM) supplemented with 2mM L-glutamine and 10% fetal bovine serum (FBS).
The DNA was prepared for transfection by sterile ethanol precipitation. Following ethanol precipitation, the DNA pellet was dried inside a tissue culture hood. The pellet was then resuspended in 450 μL of sterile water and 50 μL of 2.5 M CaCl2. Ten μg of DNA were used per 10 cm dish. While gently swirling the DNA mixture, 500 μL of sterile 2x BBS (50mM N,N-bis 2-hydroxyethyl-2-aminoethane sulfonic acid, 280mM NaCl2 and 1.5mM Na.HPO4) was added. The BBS/DNA-CaCl2 solution was allowed to form a precipitate by sitting at room temperature for 10-20 minutes.
The solution was then gently mixed to ensure adequate suspension of the precipitate and then added dropwise into the 10 cm dish of cells. The plate was gently swirled to distribute contents evenly. After a 12-16 hour incubation, the medium was carefully removed, and the cells were washed once with 5 ml of PBS (without Ca2+ or Mg2+) followed by the addition of 10ml of EMEM supplemented with 2mM L-glutamine and 10% FBS.
Following an overnight incubation, the medium was removed, and the cells were carefully washed once with 5 ml of PBS (without Ca2+ or Mg2+). To initiate selection, 10 ml of fresh EMEM with L-glutamine supplemented with 2 mM L-glutamine, 10% FBS and 0.4 mg/ml of geneticin (GIBCO-BRL) were added. Two or three days later, the medium was changed.
After approximately 2-3 weeks, each plate was examined under the microscope for small patches of growing cells. The patches were grown large enough to be seen as small spots on the bottom of the plate. Once at this stage, all of the medium was removed and
3 μL of trypsin was added directly to the patch of cells. By pipetting up and down several times, the patch of cells was transferred to a 24 well dish containing 1 ml of medium with geneticin. The cells were expanded from this 24 well stage to a 6 well plate or T-25 Flask. Because the 293 cells grow best in conditioned medium, cells were fed based on their rate of growth, but typically not more than once a week.

Example 2 - Comparison of transfected and untransfected 293 cells
To demonstrate the surprising results of the above transfection, and the greater accuracy obtained in using the transfected 293 cells in assays, transfected 293 cells of this invention and untransfected 293 cells were seeded at the same cell density (100,000 per well) into 24- well plastic tissue culture dishes. These cells were allowed to grow for two days before testing. Cell growth appeared to be equivalent.
The same biochemical assay was performed on the transfected and untransfected cells.
The presence of macrophage scavenger receptors was confirmed by incubating transfected 293 cells with 1 5[I]-acetylated LDL at a concentration of approximately 5 μg/ml (specific activity -100-300 cpm/ng protein) for 5 hours at 37°C, essentially as described in J. Ashkenas et al, J. Lipid Res.. 34:983-1000 (1993). In replicate experiments, I25[I]-acetylated LDL binding/uptake amounted to an average of 1.75μg/mg protein (n=76). Where it has been possible to measure 125[I]-acetylated LDL binding/uptake to untransfected 293 cells, the average was 0.20 μg/mg protein (n=6). After the assays were performed on the cells, they were dissolved in 0.1 M NaOH, and aliquots were used to determine total protein concentration by the Pierce BCA assay with bovine serum albumin as the standard. In an attempt to keep as many untranfected cells as possible attached to the culture dished, the untransfected cells were washed only twice, while the transfected cells were washed seven times as per the procedure cited above.
Superior attachment of the transfected cells was observed in a comparison of recoverable protein, with an average of 113±2.3 μg protein/well (n=24) versus the untransfected cells with an average of 21.8±4.8 μg protein well (n=12).
Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lysko, Paul G.
Elshourbagy, Nabil A.
Brawner, Mary E.

(ii) TITLE OF INVENTION: Attachment Enhanced 293 Cells

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham - Corporate Patents U.S.
(B) STREET: Mailcode - UW2220, 709 Swedeland Road
(C) CITY: King of Prussia
(D) STATE: Pennsylvania
(E) COUNTRY: U.S.A.
(F) ZIP: 19406-5090

(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jervis, Herbert H.
(B) REGISTRATION NUMBER: 31,171
(C) REFERENCE/DOCKET NUMBER: SBC-P50338

(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (610) 270-5019
(B) TELEFAX: (610) 270-5090 (2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2028 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: cDNA to mRNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 47..1402

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AGAGAAGTGG ATAAATCAGT GCTGCTTTCT TTAGGACGAA AGAAGT ATG GAG CAG 55
Met Glu Gin
1

TGG GAT CAC TTT CAC AAT CAA CAG GAG GAC ACT GAT AGC TGC TCC GAA 103 Trp Asp His Phe His Asn Gin Gin Glu Asp Thr Asp Ser Cys Ser Glu
5 10 15

TCT GTG AAA TTT GAT GCT CGC TCA ATG ACA GCT TTG CTT CCT CCG AAT 151 Ser Val Lys Phe Asp Ala Arg Ser Met Thr Ala Leu Leu Pro Pro Asn
20 25 30 35

CCT AAA AAC AGC CCT TCC CTT CAA GAG AAA CTG AAG TCC TTC AAA GCT 199 Pro Lys Asn Ser Pro Ser Leu Gin Glu Lys Leu Lys Ser Phe Lys Ala
40 45 50

GCA CTG ATT GCC CTT TAC CTC CTC GTG TTT GCA GTT CTC ATC CCT CTC 247 Ala Leu lie Ala Leu Tyr Leu Leu Val Phe Ala Val Leu lie Pro Leu
55 60 65

ATT GGA ATA GTG GCA GCT CAA CTC CTG AAG TGG GAA ACG AAG AAT TGC 295 lie Gly lie Val Ala Ala Gin Leu Leu Lys Trp Glu Thr Lys Asn Cys
70 75 80

TCA GTT AGT TCA ACT AAT GCA AAT GAT ATA ACT CAA AGT CTC ACG GGA 343 Ser Val Ser Ser Thr Asn Ala Asn Asp lie Thr Gin Ser Leu Thr Gly
85 90 95 AAA GGA AAT GAC AGC GAA GAG GAA ATG AGA TTT CAA GAA GTC TTT ATG 391 Lys Gly Asn Asp Ser Glu Glu Glu Met Arg Phe Gin Glu Val Phe Met
100 105 110 115

GAA CAC ATG AGC AAC ATG GAG AAG AGA ATC CAG CAT ATT TTA GAC ATG 439 Glu His Met Ser Asn Met Glu Lys Arg lie Gin His lie Leu Asp Met
120 125 130

GAA GCC AAC CTC ATG GAC ACA GAG CAT TTC CAA AAT TTC AGC ATG ACA 487 Glu Ala Asn Leu Met Asp Thr Glu His Phe Gin Asn Phe Ser Met Thr
135 140 145

ACT GAT CAA AGA TTT AAT GAC ATT CTT CTG CAG CTA AGT ACC TTG TTT 535 Thr Asp Gin Arg Phe Asn Asp lie Leu Leu Gin Leu Ser Thr Leu Phe
150 155 160

TCC TCA GTC CAG GGA CAT GGG AAT GCA ATA GAT GAA ATC TCC AAG TCC 583 Ser Ser Val Gin Gly His Gly Asn Ala lie Asp Glu lie Ser Lys Ser
165 170 175

TTA ATA AGT TTG AAT ACC ACA TTG CTT GAT TTG CAG CTC AAC ATA GAA 631 Leu lie Ser Leu Asn Thr Thr Leu Leu Asp Leu Gin Leu Asn lie Glu
180 185 190 195

AAT CTG AAT GGC AAA ATC CAA GAG AAT ACC TTC AAA CAA CAA GAG GAA 679 Asn Leu Asn Gly Lys lie Gin Glu Asn Thr Phe Lys Gin Gin Glu Glu
200 205 210

ATC AGT AAA TTA GAG GAG CGT GTT TAC AAT GTA TCA GCA GAA ATT ATG 727 lie Ser Lys Leu Glu Glu Arg Val Tyr Asn Val Ser Ala Glu lie Met
215 220 225

GCT ATG AAA GAA GAA CAA GTG CAT TTG GAA CAG GAA ATA AAA GGA GAA 775 Ala Met Lys Glu Glu Gin Val His Leu Glu Gin Glu lie Lys Gly Glu
230 235 240

GTG AAA GTA CTG AAT AAC ATC ACT AAT GAT CTC AGA CTG AAA GAT TGG 823 Val Lys Val Leu Asn Asn lie Thr Asn Asp Leu Arg Leu Lys Asp Trp
245 250 255 GAA CAT TCT CAG ACC TTG AGA AAT ATC ACT TTA ATT CAA GGT CCT CCT 871 Glu His Ser Gin Thr Leu Arg Asn lie Thr Leu lie Gin Gly Pro Pro
260 265 270 275

GGA CCC CCG GGT GAA AAA GGA GAT CGA GGT CCC ACT GGA GAA AGT GGT 919 Gly Pro Pro Gly Glu Lys Gly Asp Arg Gly Pro Thr Gly Glu Ser Gly
280 285 290

CCA CGA GGA TTT CCA GGT CCA ATA GGT CCT CCG GGT CTT AAA GGT GAT 967 Pro Arg Gly Phe Pro Gly Pro lie Gly Pro Pro Gly Leu Lys Gly Asp
295 300 305

CGG GGA GCA ATT GGC TTT CCT GGA AGT CGA GGA CTC CCA GGA TAT GCC 1015 Arg Gly Ala lie Gly Phe Pro Gly Ser Arg Gly Leu Pro Gly Tyr Ala
310 315 320

GGA AGG CCA GGA AAT TCT GGA CCA AAA GGC CAG AAA GGG GAA AAG GGG 1063 Gly Arg Pro Gly Asn Ser Gly Pro Lys Gly Gin Lys Gly Glu Lys Gly
325 330 335

AGT GGA AAC ACA TTA ACT CCA TTT ACG AAA GTT CGA CTG GTC GGT GGG 1111 Ser Gly Asn Thr Leu Thr Pro Phe Thr Lys Val Arg Leu Val Gly Gly
340 345 350 355

AGC GGC CCT CAC GAG GGG AGA GTG GAG ATA CTC CAC AGC GGC CAG TGG 1159 Ser Gly Pro His Glu Gly Arg Val Glu lie Leu His Ser Gly Gin Trp
360 365 370

GGT ACA ATT TGT GAC GAT CGC TGG GAA GTG CGC GTT GGA CAG GTC GTC 1207 Gly Thr lie Cys Asp Asp Arg Trp Glu Val Arg Val Gly Gin Val Val
375 380 385

TGT AGG AGC TTG GGA TAC CCA GGT GTT CAA GCC GTG CAC AAG GCA GCT 1255 Cys Arg Ser Leu Gly Tyr Pro Gly Val Gin Ala Val His Lys Ala Ala
390 395 400

CAC TTT GGA CAA GGT ACT GGT CCA ATA TGG CTG AAT GAA GTG TTT TGT 1303 His Phe Gly Gin Gly Thr Gly Pro lie Trp Leu Asn Glu Val Phe Cys
405 410 415

TTT GGG AGA GAA TCA TCT ATT GAA GAA TGT AAA ATT CGG CAA TGG GGG 1351 Phe Gly Arg Glu Ser Ser lie Glu Glu Cys Lys lie Arg Gin Trp Gly
420 425 430 435 ACA AGA GCC TGT TCA CAT TCT GAA GAT GCT GGA GTC ACT TGC ACT TTA 1399 Thr Arg Ala Cys Ser His Ser Glu Asp Ala Gly Val Thr Cys Thr Leu
440 445 450

TAA TGCATCATAT TTTCATTCAC AACTATGAAA TCGCTGCTCA AAAATGATTT 1452

TATTACCTTG TTCCTGTAAA ATCCATTTAA TCAATATTTA AGAGATTAAG AATATTGCCC 1512

AAATAATATT TTAGATTACA GGATTAATAT ATTGAACACC TTCATGCTTA CTATTTTATG 1572

TCTATATTTA AATCATTTTA ACTTCTATAG GTTTTTAAAT GGAATTTTCT AATATAATGA 1632

CTTATATGCT GAATTGAACA TTTTGAAGTT TATAGCTTCC AGATTACAAA GGCCAAGGGT 1692

AATAGAAATG CATACCAGTA ATTGGCTCCA ATTCATAATA TGTTCACCAG GAGATTACAA 1752

TTTTTTGCTC TTCTTGTCTT TGTAATCTAT TTAGTTGATT TTAATTACTT TCTGAATAAC 1812

GGAAGGGATC AGAAGATATC TTTTGTGCCT AGATTGCAAA ATCTCCAATC CACACATATT 1872

GTTTTAAAAT AAGAATGTTA TCCAACTATT AAGATATCTC AATGTGCAAT AACTTGTGTA 1932

TTAGATATCA ATGTTAATGA TATGTCTTGG CCACTATGGA CCAGGGAGCT TATTTTTCTT 1992

GTCATGTACT GACAACTGTT TAATTGAATC ATGAAG 2028

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 452 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Glu Gin Trp Asp His Phe His Asn Gin Gin Glu Asp Thr Asp Ser Cys Ser Glu Ser Val Lys Phe Asp Ala Arg Ser Met Thr Ala Leu Leu 20 25 30

Pro Pro Asn Pro Lys Asn Ser Pro Ser Leu Gin Glu Lys Leu Lys Ser 35 40 45

Phe Lys Ala Ala Leu lie Ala Leu Tyr Leu Leu Val Phe Ala Val Leu 50 55 60

lie Pro Leu lie Gly lie Val Ala Ala Gin Leu Leu Lys Trp Glu Thr 65 70 75 80

Lys Asn Cys Ser Val Ser Ser Thr Asn Ala Asn Asp lie Thr Gin Ser
85 90 95

Leu Thr Gly Lys Gly Asn Asp Ser Glu Glu Glu Met Arg Phe Gin Glu 100 105 110

Val Phe Met Glu His Met Ser Asn Met Glu Lys Arg lie Gin His lie 115 120 125

Leu Asp Met Glu Ala Asn Leu Met Asp Thr Glu His Phe Gin Asn Phe 130 135 140

Ser Met Thr Thr Asp Gin Arg Phe Asn Asp lie Leu Leu Gin Leu Ser 145 150 155 160

Thr Leu Phe Ser Ser Val Gin Gly His Gly Asn Ala lie Asp Glu lie
165 170 175

Ser Lys Ser Leu lie Ser Leu Asn Thr Thr Leu Leu Asp Leu Gin Leu 180 185 190

Asn lie Glu Asn Leu Asn Gly Lys lie Gin Glu Asn Thr Phe Lys Gin 195 200 205

Gin Glu Glu lie Ser Lys Leu Glu Glu Arg Val Tyr Asn Val Ser Ala 210 215 220

Glu He Met Ala Met Lys Glu Glu Gin Val His Leu Glu Gin Glu He 225 230 235 240 Lys Gly Glu Val Lys Val Leu Asn Asn He Thr Asn Asp Leu Arg Leu
245 250 255

Lys Asp Trp Glu His Ser Gin Thr Leu Arg Asn He Thr Leu He Gin 260 265 270

Gly Pro Pro Gly Pro Pro Gly Glu Lys Gly Asp Arg Gly Pro Thr Gly 275 280 285

Glu Ser Gly Pro Arg Gly Phe Pro Gly Pro He Gly Pro Pro Gly Leu 290 295 300

Lys Gly Asp Arg Gly Ala He Gly Phe Pro Gly Ser Arg Gly Leu Pro 305 310 315 320

Gly Tyr Ala Gly Arg Pro Gly Asn Ser Gly Pro Lys Gly Gin Lys Gly
325 330 335

Glu Lys Gly Ser Gly Asn Thr Leu Thr Pro Phe Thr Lys Val Arg Leu 340 345 350

Val Gly Gly Ser Gly Pro His Glu Gly Arg Val Glu He Leu His Ser 355 360 365

Gly Gin Trp Gly Thr He Cys Asp Asp Arg Trp Glu Val Arg Val Gly 370 375 380

Gin Val Val Cys Arg Ser Leu Gly Tyr Pro Gly Val Gin Ala Val His 385 390 395 400

Lys Ala Ala His Phe Gly Gin Gly Thr Gly Pro He Trp Leu Asn Glu
405 410 415

Val Phe Cys Phe Gly Arg Glu Ser Ser He Glu Glu Cys Lys He Arg 420 425 430

Gin Trp Gly Thr Arg Ala Cys Ser His Ser Glu Asp Ala Gly Val Thr 435 440 445

Cys Thr Leu *
450 ( 2 ) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1367 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: cDNA to mRNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 67..1143

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TAGGTTTCAA TTGTAAAGAG AGAGAAGTGG ATAAATCAGT GCTGCTTTCT TTAGGACGAA 60

AGAAGT ATG GAG CAG TGG GAT CAC TTT CAC AAT CAA CAG GAG GAC ACT 108 Met Glu Gin Trp Asp His Phe His Asn Gin Gin Glu Asp Thr
1 5 10

GAT AGC TGC TCC GAA TCT GTG AAA TTT GAT GCT CGC TCA ATG ACA GCT 156 Asp Ser Cys Ser Glu Ser Val Lys Phe Asp Ala Arg Ser Met Thr Ala
15 20 25 30

TTG CTT CCT CCG AAT CCT AAA AAC AGC CCT TCC CTT CAA GAG AAA CTG 204 Leu Leu Pro Pro Asn Pro Lys Asn Ser Pro Ser Leu Gin Glu Lys Leu
35 40 45

AAG TCC TTC AAA GCT GCA CTG ATT GCC CTT TAC CTC CTC GTG TTT GCA 252 Lys Ser Phe Lys Ala Ala Leu He Ala Leu Tyr Leu Leu Val Phe Ala
50 55 60

GTT CTC ATC CCT CTC ATT GGA ATA GTG GCA GCT CAA CTC CTG AAG TGG 300 Val Leu He Pro Leu He Gly He Val Ala Ala Gin Leu Leu Lys Trp
65 70 75

GAA ACG AAG AAT TGC TCA GTT AGT TCA ACT AAT GCA AAT GAT ATA ACT 348 Glu Thr Lys Asn Cys Ser Val Ser Ser Thr Asn Ala Asn Asp He Thr
80 85 90 CAA AGT CTC ACG GGA AAA GGA AAT GAC AGC GAA GAG GAA ATG AGA TTT 396 Gin Ser Leu Thr Gly Lys Gly Asn Asp Ser Glu Glu Glu Met Arg Phe
95 100 105 110

CAA GAA GTC TTT ATG GAA CAC ATG AGC AAC ATG GAG AAG AGA ATC CAG 444 Gin Glu Val Phe Met Glu His Met Ser Asn Met Glu Lys Arg He Gin
115 120 125

CAT ATT TTA GAC ATG GAA GCC AAC CTC ATG GAC ACA GAG CAT TTC CAA 492 His He Leu Asp Met Glu Ala Asn Leu Met Asp Thr Glu His Phe Gin
130 135 140

AAT TTC AGC ATG ACA ACT GAT CAA AGA TTT AAT GAC ATT CTT CTG CAG 540 Asn Phe Ser Met Thr Thr Asp Gin Arg Phe Asn Asp He Leu Leu Gin
145 150 155

CTA AGT ACC TTG TTT TCC TCA GTC CAG GGA CAT GGG AAT GCA ATA GAT 588 Leu Ser Thr Leu Phe Ser Ser Val Gin Gly His Gly Asn Ala He Asp
160 165 170

GAA ATC TCC AAG TCC TTA ATA AGT TTG AAT ACC ACA TTG CTT GAT TTG 636 Glu He Ser Lys Ser Leu He Ser Leu Asn Thr Thr Leu Leu Asp Leu
175 180 185 190

CAG CTC AAC ATA GAA AAT CTG AAT GGC AAA ATC CAA GAG AAT ACC TTC 684 Gin Leu Asn He Glu Asn Leu Asn Gly Lys He Gin Glu Asn Thr Phe
195 200 205

AAA CAA CAA GAG GAA ATC AGT AAA TTA GAG GAG CGT GTT TAC AAT GTA 732 Lys Gin Gin Glu Glu He Ser Lys Leu Glu Glu Arg Val Tyr Asn Val
210 215 220

TCA GCA GAA ATT ATG GCT ATG AAA GAA GAA CAA GTG CAT TTG GAA CAG 780 Ser Ala Glu He Met Ala Met Lys Glu Glu Gin Val His Leu Glu Gin
225 230 235

GAA ATA AAA GGA GAA GTG AAA GTA CTG AAT AAC ATC ACT AAT GAT CTC 828 Glu He Lys Gly Glu Val Lys Val Leu Asn Asn He Thr Asn Asp Leu
240 245 250

AGA CTG AAA GAT TGG GAA CAT TCT CAG ACC TTG AGA AAT ATC ACT TTA 876 Arg Leu Lys Asp Trp Glu His Ser Gin Thr Leu Arg Asn He Thr Leu
255 260 265 270 ATT CAA GGT CCT CCT GGA CCC CCG GGT GAA AAA GGA GAT CGA GGT CCC 924 He Gin Gly Pro Pro Gly Pro Pro Gly Glu Lys Gly Asp Arg Gly Pro
275 280 285

ACT GGA GAA AGT GGT CCA CGA GGA TTT CCA GGT CCA ATA GGT CCT CCG 972 Thr Gly Glu Ser Gly Pro Arg Gly Phe Pro Gly Pro He Gly Pro Pro
290 295 300

GGT CTT AAA GGT GAT CGG GGA GCA ATT GGC TTT CCT GGA AGT CGA GGA 1020 Gly Leu Lys Gly Asp Arg Gly Ala He Gly Phe Pro Gly Ser Arg Gly
305 310 315

CTC CCA GGA TAT GCC GGA AGG CCA GGA AAT TCT GGA CCA AAA GGC CAG 1068 Leu Pro Gly Tyr Ala Gly Arg Pro Gly Asn Ser Gly Pro Lys Gly Gin
320 325 330

AAA GGG GAA AAG GGG AGT GGA AAC ACA TTA AGA CCA GTA CAA CTC ACT 1116 Lys Gly Glu Lys Gly Ser Gly Asn Thr Leu Arg Pro Val Gin Leu Thr
335 340 345 350

GAT CAT ATT AGG GCA GGG CCC TCT TAA GATCAGGTGG GTTGGGCGGG 1163

Asp His He Arg Ala Gly Pro Ser *
355

ACATCCTCTG CTACCATCTC ATTAAAAGGC CCTTCACCTC TGGACAAGTC ATCTGCAACA 1223

ACTGACTTCC AAGATCCTTT TGTGACTCCT CCAAATGACT TTGGTTCCCG TGTTGTACCT 1283

GACTTCCACA TGGCCTTCTC TCCTGGTCCC TGGTGCTGTT TGGGCCTCTG CTCCCATGCT 1343

CATACCTCTT CTTACTCCAA TTAC 1367

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Glu Gin Trp Asp His Phe His Asn Gin Gin Glu Asp Thr Asp Ser 1 5 10 15

Cys Ser Glu Ser Val Lys Phe Asp Ala Arg Ser Met Thr Ala Leu Leu 20 25 30

Pro Pro Asn Pro Lys Asn Ser Pro Ser Leu Gin Glu Lys Leu Lys Ser 35 40 45

Phe Lys Ala Ala Leu He Ala Leu Tyr Leu Leu Val Phe Ala Val Leu 50 55 60

He Pro Leu He Gly He Val Ala Ala Gin Leu Leu Lys Trp Glu Thr 65 70 75 80

Lys Asn Cys Ser Val Ser Ser Thr Asn Ala Asn Asp He Thr Gin Ser
85 90 95

Leu Thr Gly Lys Gly Asn Asp Ser Glu Glu Glu Met Arg Phe Gin Glu 100 105 110

Val Phe Met Glu His Met Ser Asn Met Glu Lys Arg He Gin His He 115 120 125

Leu Asp Met Glu Ala Asn Leu Met Asp Thr Glu His Phe Gin Asn Phe 130 135 140

Ser Met Thr Thr Asp Gin Arg Phe Asn Asp He Leu Leu Gin Leu Ser 145 150 155 160

Thr Leu Phe Ser Ser Val Gin Gly His Gly Asn Ala He Asp Glu He
165 170 175

Ser Lys Ser Leu He Ser Leu Asn Thr Thr Leu Leu Asp Leu Gin Leu 180 185 190

Asn He Glu Asn Leu Asn Gly Lys He Gin Glu Asn Thr Phe Lys Gin 195 200 205

Gin Glu Glu lie Ser Lys Leu Glu Glu Arg Val Tyr Asn Val Ser Ala 210 215 220 Glu He Met Ala Met Lys Glu Glu Gin Val His Leu Glu Gin Glu He 225 230 235 240

Lys Gly Glu Val Lys Val Leu Asn Asn He Thr Asn Asp Leu Arg Leu
245 250 255

Lys Asp Trp Glu His Ser Gin Thr Leu Arg Asn He Thr Leu He Gin 260 265 270

Gly Pro Pro Gly Pro Pro Gly Glu Lys Gly Asp Arg Gly Pro Thr Gly 275 280 285

Glu Ser Gly Pro Arg Gly Phe Pro Gly Pro He Gly Pro Pro Gly Leu 290 295 300

Lys Gly Asp Arg Gly Ala He Gly Phe Pro Gly Ser Arg Gly Leu Pro 305 310 315 320

Gly Tyr Ala Gly Arg Pro Gly Asn Ser Gly Pro Lys Gly Gin Lys Gly
325 330 335

Glu Lys Gly Ser Gly Asn Thr Leu Arg Pro Val Gin Leu Thr Asp His 340 345 350

He Arg Ala Gly Pro Ser
355