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Isolation and Characterization of Human Prorenin Secreted from Murine Cells Transformed with a Bovine Papilloma Virus–Preprorenin Expression Vector

Abstract

We report the construction of a plasmid–based expression vector that carries the murine metallothionein gene promoter, the human preprorenin gene, the Tn5 phosphotransferase gene, and a complete bovine papilloma virus genome. Murine cells transformed with this vector constitutively secrete high levels of human prorenin as determined by immunoprecipitation of culture media with anti–human renin antibody and activity assays. An immunoaffinity system for the isolation of human prorenin from serum–free media, or media containing serum, was developed. Purified human prorenin is stable for months and is fully activated to enzymatically mature renin by limited tryptic digestion. This is the first example of a recombinant system leading to the isolation of research quantities of highly pure and fully activatable human prorenin.

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References

  1. Ondetti, M.A. and Cushman, D.W. 1982. Enzymes of the renin-angiotensin system and their inhibitors. Annu. Rev. Biochem. 51: 238–308.

    Article  Google Scholar 

  2. Slater, E.E. 1981. in Methods in Enzymology 80: 427–442.

    Article  CAS  Google Scholar 

  3. Nakanishi, S., Kitamura, N. and Ohkubo, H. 1985. Structure, regulation, and evolution of the genes for the renin-angiotensin and the kallekrien-kinin systems. Bio/Technology 3: 1089–1098.

    Google Scholar 

  4. Navar, L.G. 1986. Physiological role of the intrarenal renin-angiotensin system. Fed. Proc. 45: 1411–1453.

    CAS  PubMed  Google Scholar 

  5. Arakawa, K., Nakatani, M., Minohara, A. and Nakamura, M. 1967. Isolation and amino acid composition of human angiotensin I. Biochem. J. 104: 900–906.

    Article  CAS  Google Scholar 

  6. Tewksbury, D.A., Dart, R.A. and Travis, J.A. 1981. The amino terminal amino acid sequence of human angiotensinogen. Biochem. Biophys. Res. Commun. 99: 1311–1315.

    Article  CAS  Google Scholar 

  7. Oparil, S. and Haber, E. 1974. The renin-angiotensin system. New Eng. J. Med. 291: 389–401.

    Article  CAS  Google Scholar 

  8. Takahashi, S., Miura, R., Yutani, C. and Miyake, Y. 1986. Purification and characterization of human kidney renin. J. Biochem. 99: 307–310.

    Article  CAS  Google Scholar 

  9. Day, R.P. and Morris, B.J. 1979. Properties of inactive renin in human plasma. Clin. and Expt. Pharmacol. and Physiol. 6: 611–624.

    Article  CAS  Google Scholar 

  10. Sealey, J.E., Atlas, S.A. and Laragh, J.H. 1980. Prorenin and other large molecular weight forms of renin. Endocrine Reviews 1: 365–391.

    Article  CAS  Google Scholar 

  11. Leckie, B.J. 1981. Inactive renin: An attempt at a perspective. Clinical Science 60: 119–130.

    Article  CAS  Google Scholar 

  12. Morris, B.J. 1978. Properties of the activation by pepsin of inactive renin in human amniotic fluid. Biochem. Biophys. Acta 527: 86–97.

    CAS  PubMed  Google Scholar 

  13. Atlas, S.A., Laragh, J.H., Sealey, J.E. and Hesson, T.E. 1980. An inactive, prorenin-like substance in human kidney and plasma. Clinical Science 59: 29s–33s.

    Article  CAS  Google Scholar 

  14. Morris, B.J. 1978. Activation of human inactive (“Pro-”) renin by cathepsin D and pepsin. J. Clin. Endocrinol and Metab. 46: 153–157.

    Article  CAS  Google Scholar 

  15. Kim, S., Hirose, S., Miyazaki, H., Ueno, N., Higashimori, K., Morinaga, S., Kimura, T., and Murakami, K. 1985. Identification of plasma inactive renin as prorenin with a site-directed antibody. Biochem. Biophys. Res. Commun. 126: 641–645.

    Article  CAS  Google Scholar 

  16. Hirose, S., Kim, S., Miyazaki, H., Park, Y. and Murakami, K. 1985. In vitro biosynthesis of human renin and identification of plasma inactive renin as an activation intermediate. J. Biol. Chem. 260: 16400–16405.

    CAS  PubMed  Google Scholar 

  17. Hsueh, W.A., Du, Y.S., Shinagawa, T., Tam, H., Ponte, P.A., Baxter, J.D., Shine, J. and Fritz, L.C. 1986. Biochemical similarity of expressed human prorenin and native inactive renin. Hypertension 8:II-78–83.

    Google Scholar 

  18. Imai, T., Miyazaki, H., Hirose, S., Hori, H., Hayashi, T., Kageyame, R., Ohkubo, H., Nakanishi, S. and Murakami, K. 1983. Cloning and sequence analysis of cDNA for human renin precursor. Proc. Natl. Acad. Sci. USA 80: 7405–7409.

    Article  CAS  Google Scholar 

  19. Poorman, R.A., Palermo, D.P., Post, L.E., Murakami, K., Kinner, J.H., Smith, C.W., Reardon, I. and Heinrikson, R.L. 1986. Isolation and characterization of native human renin derived from Chinese hamster ovary cells. Proteins: Structure, Function, and Genetics 1: 139–145.

    Article  CAS  Google Scholar 

  20. Weighous, T.F., Cornette, J.C., Sharma, S.K. and Tarpley, W.G. 1986. Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector. Gene 45: 121–129.

    Article  CAS  Google Scholar 

  21. Law, M.F., Byrne, J.C. and Howley, P.M. 1983. A stable bovine papilloma virus hybrid plasmid that expresses a dominant selective trait. Mol. Cell. Biol. 3: 2110–2115.

    Article  CAS  Google Scholar 

  22. DiMaio, D., Treisman, R. and Maniatis, T. 1982. Bovine papilloma virus vector that propagates as a plasmid in both mouse and bacterial cells. Proc. Natl. Acad. Sci USA 79: 4030–4034.

    Article  CAS  Google Scholar 

  23. Zinn, K., Mellon, P., Ptashne, M. and Maniatis, T. 1982. Regulated expression of an extrachromosomally human β-interferon gene in mouse cells. Proc. Natl. Acad. Sci. USA 79: 4897–4901.

    Article  CAS  Google Scholar 

  24. Mitriani-Rosenbaum, S., Mariteaux, L., Mory, Y., Revel, M. and Howley, P.M. 1983. Inducible expression of the human interferon β1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells. Mol. Cell Biol. 3: 233–240.

    Article  Google Scholar 

  25. Hsiung, N., Fitts, R., Wilson, S., Milne, A. and Hamer, D. 1984. Efficient production of hepatitis β surface antigen using a bovine papilloma virus-metallothionein vector. Jour. of Mol. and Appl. Genet. 2: 497–506.

    CAS  Google Scholar 

  26. Slater, E.E. and Stout, H.V. 1981. Pure human renin. J. Biol. Chem. 256: 8164–8171.

    CAS  PubMed  Google Scholar 

  27. Yokosawa, H., Holladay, L.A., Inagami, T., Hals, E. and Murakami, K. 1980. Human renal renin: complete purification and characterization. J. Biol. Chem. 255: 3498–3502.

    CAS  PubMed  Google Scholar 

  28. Hunkapiller, M. 1985. PTH amino acid analysis. Appl. Biosystems User Bull. 14: 1–27.

    Google Scholar 

  29. Fritz, L.C., Arfsten, A.E., Dzan, V.J., Atlas, S.A., Baxter, J.D., Fiddes, J.C., Shine, J., Cofer, C.L., Kushner, P. and Ponte, P.A. 1986. Characterization of human prorenin expressed in mammalian cells from cloned cDNA. Proc. Natl. Acad. Sci. USA 83: 4114–4118.

    Article  CAS  Google Scholar 

  30. Sharma, S.K. and Hopkins, T.R. 1981. Recent developments in the activation process of bovine chymotrypsinogen A. Bioorganic Chem. 10: 357–374.

    Article  CAS  Google Scholar 

  31. Inagami, T. and Murakami, K. 1980. Prorenin. Biomedical Res. 1: 456–475.

    Article  CAS  Google Scholar 

  32. Lumbers, E.R. 1971. Activation of renin in human amniotic fluid by low pH. Enzymologia 40: 329–336.

    CAS  PubMed  Google Scholar 

  33. Derkx, F.H.M., Bouma, B.N. and Schalekamp, M.A.D.H. 1984. Prorenin-renin conversion by the contact activation system in human plasma: Role of plasma protease inhibitors. J. Lab. Clin. Med. 103: 560–573.

    CAS  PubMed  Google Scholar 

  34. Derkx, F.H.M., Schalekamp, M.P.A. and Schalekamp, M.A.D.H. 1987. Two-step prorenin-renin conversion. Isolation of an intermediary form of activated prorenin. J. Biol. Chem. 262: 2472–2477.

    CAS  PubMed  Google Scholar 

  35. Akahane, K., Umeyama, H., Nakagawa, S., Moriguchi, I., Hirose, S., Iizuka, I. and Murakami, K. 1985. Three-dimensional structure of human renin. Hypertension 7: 3–12.

    Article  CAS  Google Scholar 

  36. Weighous, T.F. and Tarpley, W.G. 1987. A bacteriophage transcription terminator permits the cloning of a mammalian expression vector carrying the human preprorenin gene in E. coli. Biochem. Biophys. Res. Comm., 143: 593–599.

    Article  CAS  Google Scholar 

  37. Guarneros, G., Montanez, C., Hernandez, T. and Court, D. 1982. Posttranscriptional control of bacteriophage λint gene expression from a site distal to the gene. Proc. Natl. Acad. Sci. USA 79: 238–242.

    Article  CAS  Google Scholar 

  38. Schmeissner, U., McKenney, K., Rosenberg, M. and Court, D. 1984. Transcription terminator involved in the expression of the int gene of phage lambda. Gene 28: 343–350.

    Article  CAS  Google Scholar 

  39. March, S.C., Parikh, I. and Cuatrecasas, P. 1974. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60: 149–152.

    Article  CAS  Google Scholar 

  40. Cuatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245: 3059–3065.

    CAS  PubMed  Google Scholar 

  41. Cornette, J.C., Evans, D.B., Furlong, A.M. and Sharma, S.K. 1987. Renin activity determination using human plasma as a substrate. Anal. Biochem. 162: 93–99.

    Article  Google Scholar 

  42. Morse, L.S., Pereira, L., Roizman, B. and Shaffer, P.A. 1978. Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1X-HSV-2 recombinants. J. Virol. 26: 389–410.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Merril, C.R., Goldman, D., Sedman, S.A. and Ebert, M.H. 1981. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211: 1437–1438.

    Article  CAS  Google Scholar 

  44. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications., Proc. Natl. Acad. Sci. USA 76: 4350–4354.

    Article  CAS  Google Scholar 

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Evans, D., Weighous, T., Cornette, J. et al. Isolation and Characterization of Human Prorenin Secreted from Murine Cells Transformed with a Bovine Papilloma Virus–Preprorenin Expression Vector. Nat Biotechnol 5, 705–709 (1987). https://doi.org/10.1038/nbt0787-705

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