Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 May;81(9):2708–2712. doi: 10.1073/pnas.81.9.2708

Cloning and expression of human erythropoietin cDNA in Escherichia coli.

S Lee-Huang
PMCID: PMC345139  PMID: 6371819

Abstract

Human erythropoietin (Ep) cDNA has been cloned in Escherichia coli by using pBR322 as a vector. Polyadenylylated RNA was isolated from selected human renal carcinomas with elevated Ep titers. The presence of Ep mRNA was detected by immunoprecipitation of in vitro translation products with monoclonal antibody to human Ep. Double-stranded cDNA was synthesized and inserted into the Pst I site of pBR322 by homopolymeric dG . dC tailing. The cDNA library was initially screened by colony hybridization with 32P-labeled cDNA synthesized from size-fractionated mRNA enriched in Ep message. Positive colonies were further screened immunologically by in situ radioimmunoassay with monoclonal antibody to human Ep. Three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein. These clones contain inserts of approximately 1400, 600, and 200 base pairs. Human renal Ep mRNA, of which the translation products immunoreact with anti-Ep on immunoblots, was hybrid-selected by plasmid DNA from these recombinants. Purified human Ep competes with 35S-labeled hybrid-selected translation products for antibody binding.

Full text

PDF
2708

Images in this article

2709Image
on p.2709
2709Image
on p.2709
2710Image
on p.2710
2710Image
on p.2710
2710Image
on p.2710
2711Image
on p.2711
2711Image
on p.2711
2711Image
on p.2711

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. COTES P. M., BANGHAM D. R. Bio-assay of erythropoietin in mice made polycythaemic by exposure to air at a reduced pressure. Nature. 1961 Sep 9;191:1065–1067. doi: 10.1038/1911065a0. [DOI] [PubMed] [Google Scholar]
  6. Carmena A. O., De Testa N. G., Frias F. L. Urinary erythropoietin in men subjected to acute hypoxia. Proc Soc Exp Biol Med. 1967 Jun;125(2):441–443. doi: 10.3181/00379727-125-32114. [DOI] [PubMed] [Google Scholar]
  7. Efstratiadis A., Kafatos F. C., Maxam A. M., Maniatis T. Enzymatic in vitro synthesis of globin genes. Cell. 1976 Feb;7(2):279–288. doi: 10.1016/0092-8674(76)90027-1. [DOI] [PubMed] [Google Scholar]
  8. Gordon A. S., Cooper G. W., Zanjani E. D. The kidney and erythropoiesis. Semin Hematol. 1967 Oct;4(4):337–358. [PubMed] [Google Scholar]
  9. Graber S. E., Krantz S. B. Erythropoietin and the control of red cell production. Annu Rev Med. 1978;29:51–66. doi: 10.1146/annurev.me.29.020178.000411. [DOI] [PubMed] [Google Scholar]
  10. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  13. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  15. Lee-Huang S. A new preparative method for isolation of human erythropoietin with hydrophobic interaction chromatography. Blood. 1980 Oct;56(4):620–624. [PubMed] [Google Scholar]
  16. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  17. Marks P. A., Rifkind R. A. Erythroleukemic differentiation. Annu Rev Biochem. 1978;47:419–448. doi: 10.1146/annurev.bi.47.070178.002223. [DOI] [PubMed] [Google Scholar]
  18. Miyake T., Kung C. K., Goldwasser E. Purification of human erythropoietin. J Biol Chem. 1977 Aug 10;252(15):5558–5564. [PubMed] [Google Scholar]
  19. Parnes J. R., Velan B., Felsenfeld A., Ramanathan L., Ferrini U., Appella E., Seidman J. G. Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2253–2257. doi: 10.1073/pnas.78.4.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  21. Reissmann K. R., Samorapoompichit S. Effect of erythropoietin on proliferation of erythroid stem cells in the absence of transplantable colony-forming units. Blood. 1970 Sep;36(3):287–296. [PubMed] [Google Scholar]
  22. Retzel E. F., Collett M. S., Faras A. J. Enzymatic synthesis of deoxyribonucleic acid by the avian retrovirus reverse transcriptase in vitro: optimum conditions required for transcription of large ribonucleic acid templates. Biochemistry. 1980 Feb 5;19(3):513–518. doi: 10.1021/bi00544a019. [DOI] [PubMed] [Google Scholar]
  23. Sherwood J. B., Goldwasser E. Erythropoietin production by human renal carcinoma cells in culture. Endocrinology. 1976 Aug;99(2):504–510. doi: 10.1210/endo-99-2-504. [DOI] [PubMed] [Google Scholar]
  24. Sytkowski A. J., Richie J. P., Bicknell K. A. New human renal carcinoma cell line established from a patient with erythrocytosis. Cancer Res. 1983 Mar;43(3):1415–1419. [PubMed] [Google Scholar]
  25. Thorling E. B. Paraneoplastic erythrocytosis and inappropriate erythropoietin production. A review. Scand J Haematol Suppl. 1972;17:1–166. [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Toyama K., Fujiyama N., Suzuki H., Chen T. P., Tamaoki N., Ueyama Y. Erythropoietin levels in the course of a patient with erythropoietin-producing renal cell carcinoma and transplantation of this tumor in nude mice. Blood. 1979 Jul;54(1):245–253. [PubMed] [Google Scholar]
  28. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  29. Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. WEINTRAUB A. H., GORDON A. S., BECKER E. L., CAMISCOLI J. F., CONTRERA J. F. PLASMA AND RENAL CLEARANCE OF EXOGENOUS ERYTHROPOIETIN IN THE DOG. Am J Physiol. 1964 Sep;207:523–529. doi: 10.1152/ajplegacy.1964.207.3.523. [DOI] [PubMed] [Google Scholar]
  31. Waldmann T. A., Rosse W. F., Swarm R. L. The erythropoiesis-stimulating factors produced by tumors. Ann N Y Acad Sci. 1968 Mar 29;149(1):509–515. doi: 10.1111/j.1749-6632.1968.tb15190.x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES