ATTACHMENT ENHANCED 293 CELLS

Abstract: Attachment enhanced human embryonic kidney cells, 293, are provided. These cells have been modified to contain a selected mammalian scavenger gene, which has been found to improve the ability of these cells to attach in culture. The improved cells of the invention are useful in assays in which the unmodified 293 cells could be used.

Description: 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 10 5 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 CaCl 2 . 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 NaCl 2 and 1.5mM Na.HPO 4 ) was added. The BBS/DNA-CaCl 2 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 Ca 2+ or Mg 2+ ) 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 Ca 2+ or Mg 2+ ). 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 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 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

Claims: WHAT IS CLAIMED IS: 1. Human embryonic kidney 293 cells transfected with a mammalian scavenger receptor gene, said cells demonstrating an enhanced ability to attach to a solid support. 2. The cells according to claim 1 wherein said receptor gene is a human macrophage scavenger receptor gene Type I. 3. The cells according to claim 1 wherein the receptor gene is characterized by the sequence of GenBank accession number D90187. 4. The cells according to claim 1 wherein said receptor gene is a human macrophage scavenger receptor gene Type II. 5. The cells according to claim 1 wherein the receptor gene is characterized by the sequence of GenBank accession number D90188. 6. The cells according to claim 1 wherein said receptor gene is a macrophage scavenger receptor gene of a non-human species. 7. A solid support to which is attached human embryonic kidney 293 cells transfected with a mammalian scavenger receptor gene. 8. The support according to claim 7- wherein said receptor gene is a human macrophage scavenger receptor gene Type I. 9. The support according to claim 7 wherein the receptor gene is characterized by the sequence of GenBank accession number D90187. 10. The support according to claim 7 wherein said receptor gene is a human macrophage scavenger receptor gene Type II. 11. The support according to claim 7 wherein the receptor gene is characterized by the sequence of GenBank accession number D90188. 12. The support according to claim 7 wherein said receptor gene is a macrophage scavenger receptor gene of a non-human species. 13. A method of enhancing the ability of human embryonic kidney cells to attach to a solid support comprising the steps of: (a) providing cells from a 293 cell line; and (b) transfecting the cells with a mammalian scavenger receptor gene; wherein the transfected cells are characterized by an enhanced ability to attach to said solid support. 14. The method according to claim 13 further comprising transfecting said cells with a second selected gene. 15. The method according to claim 14 wherein the selected gene is a selection marker. 16. The method according to claim 15 wherein the gene is a selectable resistance marker. 17. A method of screening a compounds for biological activity comprising the steps of: (a) providing on a solid support human embryonic kidney 293 cells co-transfected with a mammalian scavenger receptor gene and a second selected gene which encodes a protein having a biological activity; (b) measuring expression of the protein encoded by said second selected gene; (c) incubating said co-transfected 293 cells in the presence of a compound of unknown biological activity; (d) screening the cells of (c) for the ability of the compound to alter said biological activity. 18. The method according to claim 17 wherein the receptor gene is a human macrophage scavenger receptor gene Type I or Type II. 19. An improved method for screening a compound for biological activity comprising measuring in a transfected cell the expression of a protein encoded by a selected gene; incubating said transfected cell in the presence of a compound of unknown biological activity; and screening the cell for the ability of said compound to alter said biological activity, the improvement comprising employing as said transfected cell, human embryonic kidney 293 cells co-transfected with a mammalian scavenger receptor gene and said selected gene, said cells attached to a solid support. 20. An improved method for performing a biological assay on a cell attached to a solid support, wherein said assay involves at least one washing step, said improvement comprising employing as said attached cell, human embryonic kidney 293 cells co-transfected with a mammalian scavenger receptor gene. 21. An improved method for measuring the production of a protein in a cell attached to a solid support, said improvement comprising employing as said attached cell, human embryonic kidney 293 cells co-transfected with a mammalian scavenger receptor gene.

Inventors: BRAWNER MARY ELLEN, ELSHOURBAGY NABIL ABD ELSALAM, LYSKO PAUL GEORGE, BRAWNER, MARY, ELLEN, ELSHOURBAGY, NABIL, ABD, ELSALAM, LYSKO, PAUL, GEORGE
Applicants: BRAWNER MARY ELLEN, ELSHOURBAGY NABIL ABD ELSALAM, LYSKO PAUL GEORGE, SMITHKLINE BEECHAM CORP, BRAWNER, MARY, ELLEN, ELSHOURBAGY, NABIL, ABD, ELSALAM, LYSKO, PAUL, GEORGE, SMITHKLINE BEECHAM CORPORATION
Application year: 1996
Main classification: G01N33
IPC codes: C07K14, C12N5, C12N11, C12N15, C12P21, C12R1, G01N33