Cell Biology. Human erythropoietin gene: High level expression in stably ... by cotransfection of mammalian cells with a plasmid containing a selectable marker ...
Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6465-6469, September 1986 Cell Biology
Human erythropoietin gene: High level expression in stably transfected mammalian cells and chromosome localization (genomic screening/mammalian cdl expression/chromosome sorting/gene amplification)
JERRY S. POWELL*, KATHLEEN L. BERKNERt, ROGER V. LEBO*, AND JOHN W. ADAMSON* *University of Washington, Seattle, WA 98195; tZymogenetics, Inc., 2121 North 35th Street, Seattle, WA 98103; and tHoward Hughes Medical Institute and University of California, San Francisco, CA 94143
Communicated by Eloise R. Giblett, May 15, 1986
ABSTRACT The glycoprotein hormone erythropoietin plays a major role in regulating erythropoiesis and deficiencies of erythropoietin result in anemia. Detailed studies of the hormone and attempts at replacement therapy have been difficult due to the scarcity of purified material. We used a cloned human erythropoietin gene to develop stably transfected mammalian cell lines that secrete large amounts ofthe hormone with potent biological activity. These cell lines were produced by cotransfection of mammalian cells with a plasmid containing a selectable marker and plasmid constructions containing a doned human erythropoietin gene inserted next to a strong promoter. The protein secreted by these cells stimulated the proliferation and differentiation of erythroid progenitor cells and, with increased selection, several of these cell lines secrete up to 80 mg of the protein per liter of supernatant. Hybridization analysis of DNA from human chromosomes isolated by high resolution dual laser sorting provides evidence that the gene for human erythropoietin is located on human chromosome 7. Normal production of erythrocytes in man requires the secretion of erythropoietin by the kidney, apparently as the mature protein (1). In the steady state, the hormone circulates in the blood at a concentration of 10-18 milliunits (128-230 pg) per ml, and with the stimulus of severe tissue hypoxia the levels may increase as much as 1000-fold (2). The elevated hormone levels trigger proliferation and differentiation of a population of receptive progenitor cells in the bone marrow, stimulate hemoglobin synthesis in maturing erythroid cells, and accelerate release of erythrocytes from the marrow into circulation, thus increasing the erythrocyte mass and ameliorating the hypoxic conditions. Patients with deficiencies of this hormone, such as those with chronic renal failure, often suffer severe anemia. Erythropoietin is a glycoprotein of 34-38 kDa with ==40%o of its molecular size provided by carbohydrate. At least one disulfide bridge is required for activity (3, 4). However, little is known about the conformation of this hormone, and the details of its synthesis are not well understood. The isolations of genomic and cDNA clones provide opportunities to analyze the control of erythropoietin production as well as to provide sufficient quantities of material both for further study and for replacement therapy (5-7). Here we report the use of a genomic clone of human erythropoietin to develop stably transfected mammalian cell lines that secrete high levels of active erythropoietin. Furthermore, using restriction fragments of the cloned gene as probes, we have mapped the gene for human erythropoietin to chromosome 7. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
MATERIALS AND METHODS Isolation of Genomic Clones. A human genomic library in bacteriophage X (8) was screened using low stringency hybridization conditions and mixtures of oligonucleotide probes (9). Oligonucleotide mixtures were prepared using an Applied Biosystems (Foster City, CA) synthesizer and endlabeled using [32P]ATP and T4 polynucleotide kinase. The synthetic oligonucleotides were designed to correspond to portions of the amino-terminal amino acid sequence of H2N-Ala-Pro-?-Arg-Leu-Ile-Leu-Asp-Ser-Arg-Val-Leu-GluArg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-?-Ile-Thr-AspGly-Gly-Ala obtained by Yanagawa et al. (10) for the human protein purified from urine of patients with aplastic anemia. To reduce the degeneracy of the codons for the amino acid sequence of this region the codon usage rules of Grantham et al. (11) and Jaye et al. (12) were employed. These rules take into account the relatively rare occurrence of CpG dinucleotides in DNA of vertebrates and avoid, where appropriate, potential A-G mismatch pairings. At amino acid position 24 an asparagine was placed as most likely (10). For the amino acids Glu-Ala-Lys-Glu-Ala-Glu-Asn, 2 pools of 72 sequences each were synthesized to correspond to the predicted codons. Thus, one pool was TT(C/T)TC(A/G/T)GC(C/T)TC(C/T)TT(A/G/T)GCTTC for the 20-nucleotide probe and the second pool replaced a T with a C at position 18. For the amino acids Glu-Asn-lle-Thr-Asp-Gly, one pool of sequences [AGC TCC TCC ATC AGT ATT ATT T(C/T)] was constructed for the 23-nucleotide probe. Plaques that hybridized to the oligonucleotide probes were rescreened at lower density until pure. [32P]ATP was from ICN; enzymes were from New England Biolabs or Bethesda Research Laboratories. After EcoRI restriction enzyme digestion of the positive clones, insert DNA was gel-purified and ligated by standard techniques into plasmid pUC13 that had been digested with EcoRI. DNA sequence was determined by dideoxynucleotide chain termination (13) using dATP[a-355] and the 17-mer universal primer or, in selected regions, specific oligonucleotide primers. Construction of Expression Plaids Carrying Erythropoietin Gene Sequences. The plasmid expression vector pDll was derived from a previously described plasmid (14) and contained the simian virus 40 enhancer sequences and origin of replication as well as the adenovirus type 2 major late promoter and tripartite leader sequences (see Fig. 2). The Apa I fragment of human erythropoietin genomic sequences (Fig. 1) was gelpurified, and single-stranded ends were filled in by treatment with T4 polymerase. BamHI linkers were ligated to both blunt ends, and the fragment was inserted into the expression plasmid at the BamHI restriction site to form pDll-Ep. Recombinant plasmids were cloned in Escherichia coli HB101 and purified by isopycnic centrifugation in cesium Abbreviation: bp, base pair(s).
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Proc. Natl. Acad. Sci. USA 83 (1986) _
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FIG. 1. Schematic representation of the Apa I restriction fragment that contains the human erythropoietin gene
chloride. The expression plasmid pDll-Ep is approximately 6500 base pairs (bp) in length. The construction was confirmed by restriction mapping and partial dideoxynucleotide sequencing.
Transfection of Mammalian Cells. Mammalian cell lines, COS-7 (monkey kidney) and BHK (baby hamster kidney), were maintained in Dulbecco's modified essential medium containing 10% (vol/vol) fetal calf serum. Cells were passaged and, when cultures were 50-70% confluent, cells were transfected by the calcium phosphate method (15). For transient expression of cells in a 100-mm culture dish, a total of 20 ug of DNA was used as follows: 10 ,ug of plasmid containing the erythropoietin gene and 10 ,ug of carrier salmon sperm DNA. After 48 hr the supernatant was collected, centrifuged at 400 x g for 10 min to remove cells and debris, and frozen at -20°C. The cells were harvested separately. Control experiments for these transfection assays included supernatants from nontransfected cells and parallel cultures of cells transfected with plasmids containing DNA encoding other proteins, including bacterial chloramphenicol acetyltransferase and human coagulation protein, factor IX. For transfections to establish stable cell lines producing high levels oferythropoietin, either COS-7 or BHK cells were cotransfected with the pD11-Ep plasmid and pDHFRr, a plasmid containing a cDNA for dihydrofolate reductase, in a similar mammalian expression vector. The transfection procedure was modified so that 5 ,ug of pDll-Ep plasmid, 5 ,ug of pDHFRr plasmid, and 10 ,ug of carrier DNA were cotransfected. After additional incubation for 18-24 hr, various concentrations of methotrexate (10 nM to 1 mM) were added to the cultures. After incubation for several more days, viable colonies resistant to methotrexate were isolated, passaged, and screened for the presence of erythropoietin bioactivity in the supernatant. To amplify the expression of the transcriptional unit containing the erythropoietin gene and the dihydrofolate reductase (DHFR) gene, cell lines 'secreting high levels of erythropoietin were passaged several times into increasingly higher concentrations of methotrexate (16). Cell lines were considered stable if erythropoietin production remained high for more than 15 passages in the absence of methotrexate selective pressure. Assays for Erythropoietin. The in vitro assay for erythropoietin biological activity was based on the formation of erythroid colonies (from erythroid colony-forming cells) in cultures of mouse bone marrow cells in plasma clot (17). The sensitivity of this assay is about 5 milliunits per ml. The erythropoietin used as the standard for assay was a partially purified preparation from plasma from anemic sheep (Connaught, Step 3 erythropoietin, Lot 3026). Supernatants were assayed from passaged cell lines grown for 24 hr in fresh medium without methotrexate. The supernatant was diluted 1:200 with medium, and amounts between 1 and 10 IlI were added per ml of assay culture containing 2 x 105 cells, 10% (vol/vol) bovine citrated plasma, 20% (vol/vol) fetal calf serum, 1% bovine serum albumin, and 1.6% (wt/vol) beef embryo extract (GIBCO). After incubation for 36-48 hr, the plasma clots were fixed on microscope slides, stained with benzidine for hemoglobin, and erythroid colonies were counted. In the absence of added erythropoietin, no erythroid colony-forming cell-derived colonies were detected. Optimal erythroid colony growth (100-150 erythroid colony forming
sequences.
cells detected per 2 x 104 marrow cells) was observed routinely with 50 milliunits (0.64 ng) of erythropoietin per ml of culture. Selected cell lines were also assayed for in vivo erythropoietin activity in exhypoxic polycythemic mice (18). In addition, supernatants from selected cell lines were assayed for immunologically reactive erythropoietin by competitive radioimmunoassay using a polyvalent anti-human erythropoietin antiserum (19). Chromosome Mapping. Human DNA was treated with several restriction endonucleases and electrophoresed in a 1% agarose gel. Southern transfer to a nitrocellulose filter was followed by hybridization using high stringency conditions with nick-translated 32P-labeled DNA restiction fragments derived from the human erythropoietin gene. Several restriction fragments were tested to select fragments providing optimal hybridization signals and to avoid Alu repetitive sequences located in the second intron of the human erythropoietin gene. Autoradiography was performed overnight and for 10 days. The following two restriction fragments were chosen for chromosomal localization studies: a 916-bp Sma I fragment from the 5' end of the gene and an 854-bp Xmn I-Apa I fragment from the 3' end (Fig. 1). Chromosome suspensions were prepared from a lymphocyte cell line in Tris/spermine buffer and stained with the DIPI-chromomycin A3 stain pair (20). Thirty thousand chromosomes of each type were sorted directly onto a single spot of a nitrocellulose filter using a high resolution dual laser chromosome sorter (21). Thus, 21 unique fractions of human chromosomes bound to nitrocellulose filters were isolated (20). The filter-bound chromosomal DNA was denatured, neutralized, hybridized, washed, and autoradiographed using standard conditions (21). Both the 5' fragment probe and the 3' fragment probe were hybridized independently to chromosomal filter sets.
RESULTS AND DISCUSSION Isolation of Genomic Sequences. Approximately 4.8 106 bacteriophage were screened by hybridization of replicate nitrocellulose filters. Three different clones remained positive through plaque purification, and the DNA insert was characterized by restriction mapping and partial dideoxynucleotide sequencing. Two of the three clones contained apparently complete information for the erythropoietin gene. The restriction map and sequence for the Apa I fragment of these clones were essentially the same as those published by Jacobs et al. (5) for the gene for human erythropoietin. The low frequency of phage isolates containing the erythropoietin gene in this library, one in 2 x 106 bacteriophage, may be attributed to the loss of sequences during amplification procedures used to maintain this library. Southern blot hybridization of total human genomic DNA with the Apa I fragment of other restriction fragments of the erythropoietin gene indicated only a single hybridizing band with no additional regions of highly homologous DNA. This finding is consistent with the suggestion that erythropoietin exists as a single copy in the human genome. Expression of Erythropoietin Gene in Mammalian Cels. The structure of the expression plasmid pDll-Ep is shown in Fig. 2. The 2426-bp Apa I restriction fragment of the erythropoietin gene was treated with T4 DNA polymerase to produce X
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Proc. Natl. Acad. Sci. USA 83 (1986)
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FIG. 2. Diagram of the plasmid expression vector pD11-Ep. The 2426-bp Apa I fragment containing the human erythropoietin gene was treated with T4 DNA polymerase to produce blunt ends. BamHI linkers were added to the fragment, and the fragment was ligated into the unique cloning site of the plasmid vector (B, BamHI restriction site). The plasmid pD11 contains 350 bp of the adenovirus left terminus (0-1), the origin and enhancer sequences from simian virus 40 (E), the adenovirus major late promoter (MLP), the adenovirus type 2 tripartite leader (L1-3), and third leader 5' splice site (5' ss), an immunoglobulin 3' splice site (3' ss), and the late simian virus 40 polyadenylylation signal (pA) in the EcoRI (RI) restriction site of pML (24). The plasmid pD11-Ep is approximately 6500 bp in length.
blunt ends, and BamHI linkers were added to the fragment. The construct was inserted into the unique BamHI restriction site of the plasmid vector to direct transcription of the erythropoietin gene from a strong promoter. The plasmid pDll contains the adenovirus major late promoter and tripartite leader sequences that enhance translation. The inserted Apa I restriction fragment contained 58 bp of 5'-untranslated sequences followed by sequences coding for a putative 27-amino acid signal peptide, the mature protein, four intervening sequences, and 22 bp of 3' noncoding DNA sequence. This expression vector construction was chosen to optimize production of erythropoietin protein. We used the complete erythropoietin gene for expression to include potential regulatory or enhancing sequences located in introns that might contribute to erythropoietin gene expression or protein modification and secretion. The results of transfections with the erythropoietin gene alone for transient expression are shown in Table 1. The levels of erythropoietin secreted into the supernatant of either the COS-7 or BHK mammalian cell lines were -80 times higher than those reported for transient expression of a cDNA coding for erythropoietin (5, 6). The reasons for this higher expression are not clear but may relate to the use of the erythropoietin gene with intervening sequences rather than erythropoietin cDNA or to our use of a plasmid vector that is more suitable for expression. None of the control cultures, mock transfections, or cultured cells transfected with other genes had detectable erythropoietin activity. To establish stable cell lines producing large amounts of erythropoietin protein, mammalian cell lines were cotransfected with the pD11-Ep plasmid and pDHFRr plasmid containing a cDNA for dihydrofolate reductase. After transfection, the culture medium was changed to include several different concentrations of methotrexate. Cells that incorporated the dihydrofolate reductase gene would be viable in the selective medium. Thus colonies resistant to the methotrexate selection were isolated and passaged. Approximately half of the methotrexate-resistant colonies that were assayed secreted detectable erythropoietin activity. Amounts of erythropoietin activity in the cell pellets could not be determined due to the presence of significant inhibitors of the bioassay in the cell extracts. Consequently our results do not analyze the intracellular levels of erythropoietin protein but, rather, the amount of erythropoietin protein produced and secreted into the supernatant by the cell lines (Table 2). Table 1. Secretion of recombinant human erythropoietin by mammalian cells transfected with plasmid pD11-Ep for transient expression assay Erythropoietin per ml of culture Mammalian Activity, units cells (in vitro bioassay) Protein, pZg BHK 3.4 0.2 270 ± 16 COS-7 3.2 0.4 255 ± 32 Data are from three experiments for each cell type.
The recombinant erythropoietin protein secreted into the supernatant of transfected cell lines was biologically active and large amounts of hormone were secreted (up to 7000 units per ml). If the recombinant erythropoietin has a specific activity equivalent to that of natural erythropoietin (78,000 units per mg of protein), the biological assay corresponds to approximately 80 ug of erythropoietin protein per ml. The concentration of erythropoietin protein from selected cell lines also was assayed by a competitive radioimmunoassay using a polyvalent rabbit anti-human erythropoietin antiserum. The amount of protein measured by the radioimmunoassay was equivalent to the protein level estimated by the biological assay. These data indicate that the transfected cell lines expressed and secreted erythropoietin protein that was greater than 98% active. The recombinant erythropoietin produced by the transfected cells was further characterized to demonstrate that these cells were secreting authentic hormone. Supernatants from several cell lines had potent in vivo biological activity when assayed in the exhypoxic polycythemic mouse. In experiments using partially purified native erythropoietin, it has been noted that neuraminidase treatment completely abrogated erythropoietin activity when assayed in the intact animal (22). The loss of activity presumably was due to increased clearance by the liver of the desialated hormone since neuraminidase-treated erythropoietin remained fully active in vitro. The observation of potent in vivo biological activity is important primarily because it partially confirms that the transfected mammalian cell lines appropriately add carbohydrate including the terminal sialic' acids to the erythropoietin protein during posttranslational modification. In separate experiments, the activity of erythropoietin in the in vitro biological assay was neutralized by a neutralizing anti-human erythropoietin antibody added to the culture. While these data suggest that the recombinant erythropoietin produced by the transfected cell line is very similar to native erythropoietin, protein sequence determination and analyses of the carbohydrate components will be necessary to determine precisely the fidelity of expression of the erythropoietin gene and of processing the erythropoietin protein by the cell lines. The erythropoietin secreted into the supernatant of representative transfected cell lines was also assayed for proliferTable 2. Expression of recombinant erythropoietin in stably transfected mammalian cell lines Erythropoietin per ml of supernatant Activity, units (in vitro bioassay) Cell line Protein, ,ug 970 F 1.1 12.4 2500 F 3.4 32.0
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FIG. 3. Autoradiogram of sorted human chromosomes on sets of nitrocellulose filters hybridized with the 915-bp 5' probe bound by Sma I restriction sites. The 854-bp 3' probe Xmn I-Apa I gave an identical autoradiogram with an independently sorted filter set. Twelve 25-mm circular nitrocellulose filters with two chromosome spots each are displayed with the chromosome numbers on the edge of each filter adjacent to each sorted chromosome spot. Chromosome 7 produced an intense hybridization signal, mapping the gene to that chromosome.
ative effects on other bone marrow progenitor cells. Recombinant erythropoietin was assayed for its effect on a variety of progenitors from mouse and human marrow including erythroid colony-forming cells, erythroid burst-forming cells, granulocyte-macrophage precursors, and mixed-cell colonyforming cells (23). Erythroid progenitor cells exhibited a proliferation response to recombinant erythropoietin that was parallel to the dose-response relationship found with natural erythropoietin. Neither granulocyte-macrophage precursors nor mixed-cell colony-forming cells exhibited any proliferative response to the recombinant erythropoietin at concentrations up to 10 units/ml of assay cell culture. Chromosome Mapping. High resolution dual laser sorting offluorescent chromosomes was employed to sort and isolate 21 fractions of human chromosomes bound to nitrocellulose filters. The autoradiograph obtained after hybridization with the nick-translated 32P-labeled 5'-restriction fragment is shown in Fig. 3. Even after autoradiography for 1 week, only the dot blot identified as chromosome 7 DNA exhibited any hybridizing signal. A similar autoradiograph was obtained when the 3'-restriction fragment was hybridized independently. To confirm that the gene for human erythropoietin was located on chromosome 7, we subsequently hybridized the 3' probe to nitrocellulose filters of chromosomes sorted from the human fibroblast cell line GM44 that carries a reciprocal translocation of the distal portion of chromosome 7 (p21.2) exchanged for a portion of chromosome 10 (qll.21). In these experiments, the probe identified the sorted derivative chromosome 7 and not the translocated portion on derivative chromosome 10. Again, no other chromosome DNA hybridized labeled probe, even after extensive autoradiography. We have confirmed that our probes hybridize with DNA from sorted chromosome 7 by several control experiments. A probe for epidermal growth factor receptor (kindly provided by Michael Rosenfeld), which we mapped to chromosome 7 by dot-blot analysis, had been localized to chromosome 7 by somatic cell hybridization (25). This radiolabeled probe hybridized specifically to the spot for chromosome 7 in the filter set used to map erythropoietin. To date, this technique has localized 24 genes, and each assignment has been confirmed by other methods. In the present experiments, we have shown that biologically active human erythropoietin can be expressed at high levels (up to 80 mg protein per liter of supernatant) from stably transfected mammalian cell lines and that the gene for human erythropoietin is located on chromosome 7. Thus the basis is established for an abundant source of purified human erythropoietin for further biochemical and clinical studies.
We thank Barry D. Bruce for sorting the fluorescent chromosomes; Mei-Chi Cheung for hybridization of the chromosome preparations; Dr. Patrick Chou of the Department of Chemistry for preparation of the synthetic oligonucleotides; Nancy Lin and Caryl Campbell for biological assays; Dr. Joan Egrie for the radioimmunoassay determinations; Dr. Jaime Caro for performing in vivo bioassays; and Drs. Earl Davie and Kotoku Kurachi for insightful discussion. We also thank Carmen Nott for preparation of the manuscript. This work was supported in part by Research Grants HL 16919, AM 19410, and CA 31615 from the National Institutes of Health. J.S.P. is the recipient of Clinical Investigator Award AM 01418 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. 1. Jacobson, L. 0., Goldwasser, E., Fried, W. & Plzak, L. (1957) Nature (London) 179, 633-634. 2. Garcia, J. F., Ebbe, S., Hollander, L., Cutting, H. O., Miller, M. & Cronkite, E. P. (1982) J. Lab. Clin. Med. 99, 624-635. 3. Miyake, T., Kung, C. K. & Goldwasser, E. (1977) J. Biol. Chem. 252, 5558-5564. 4. Sytkowski, A. J. (1980) Biochem. Biophys. Res. Commun. 96, 143-149. 5. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S., Kaufman, J., Musfon, A., Seehra, J., Jones, S., Hewick, R., Fritsch, E., Kawakita, M., Shimizu, T. & Miyake, T. (1985) Nature (London) 313, 806-810. 6. Lin, F.-K., Suggs, S., Lin, C.-H., Browne, J. K., Smalling, R., Egrie, J. C., Chen, K. K., Fox, G. M., Martin, F., Stabinsky, Z., Badrawi, S. M., Lai, P.-H. & Goldwasser, E. (1985) Proc. NatI. Acad. Sci. USA 82, 7580-7584. 7. Powell, J. S., Segal, G. M., Berkner, K. L. & Adamson, J. W. (1985) Blood 66, 756 (abstr.). 8. Lawn, R. M., Fritsch, E. F., Parker, R. C., Blake, G. & Maniatis, T. (1978) Cell 15, 1157-1174. 9. Deryneck, R., Roberts, A. B., Winkler, M. E., Chen, E. Y. & Goeddel, D. V. (1984) Cell 38, 287-297. 10. Yanagawa, S., Kirade, K., Hideki, O., Sasaki, R., Chiba, H., Ueda, M. & Masaaki, G. S. (1984) J. Biol. Chem. 259, 2707-2710. 11. Grantham, R., Gautier, C., Gouy, M., Jacobzone, M. & Mercier, R. (1981) Nucleic Acids Res. 9, 43-59. 12. Jaye, M., de la Salle, H., Schamber, F., Balland, A., Kohli, V., Findeli, A., Tolstoshev, P. & Lecocq, J. (1983) Nucleic Acids Res. 11, 2325-2335. 13. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 14. Berkner, K. L. & Sharp, P. (1985) Nucleic Acids Res. 13, 841-857. 15. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456-467. 16. Simonsen, C. C. & Levinson, A. D. (1983) Proc. Natl. Acad. Sci. USA 80, 2495-2499.
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