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A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

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Abstract

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.

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References

  • Arakawa T, Hsu YR, Parker CG and Lai PH (1986) Role of polycationic C-terminal protein in the structure and activity of recombinant human interferon-γ. J. Biol. Chem. 261: 8534–8539.

    CAS  Google Scholar 

  • Damon DH, Lobb RR, D’Amore PA and Wagner JA (1989) Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J. Cell Physiol. 138: 221–226.

    Article  CAS  Google Scholar 

  • Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama O, Takahashi K, Miyazaki H, Hashimoto S and Daikuhara Y (1988) Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J. Clin. Invest. 81: 414–419.

    Article  CAS  Google Scholar 

  • Higashio K, Shima N, Goto M, Itagaki Y, Nagao M, Yasuda H and Morinaga T (1990) Identity of tumor cytotoxic factor human fibroblasts and hepatocyte growth factor. Biochem. Biophys. Res. Commun. 170: 397–404.

    Article  CAS  Google Scholar 

  • Kaufman RJ, Wasley LC and Dorner AJ (1988) Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J. Biol. Chem. 263: 6352–6362.

    CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  • Lobb RR (1988) Thrombin inactivates acidic fibroblast growth factor but not basic fibroblast growth factor. Biochemistry 27: 2572–2578.

    Article  CAS  Google Scholar 

  • Miyazawa K, Tsubouchi H, Naka D, Takahashi K, Okigaki M, Arakaki N, Nakayama H, Hirono S, Sakiyama O, Takahashi K, Gohda E, Daikuhara Y and Kitamura N (1989) Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163: 967–973.

    Article  CAS  Google Scholar 

  • Miyazawa K, Kitamura A, Naka D and Kitamura N (1991) An alteratively processed mRNA generated from human hepatocyte growth factor gene. Eur. J. Biochem. 197: 15–22.

    Article  CAS  Google Scholar 

  • Murakami H, Shimomura T, Nakamura T, Ohashi H, Shinohara K and Omura H (1984) Development of a basal medium for serum-free cultivation of hybridoma cells in high density. Nippon Nogeikagaku Kaishi (Japan) 58: 575–583.

    CAS  Google Scholar 

  • Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K and Shimizu S (1989) Molecular cloning and expression of human hepatocyte growth factor. Nature 342: 440–443.

    Article  CAS  Google Scholar 

  • Rijken DC and Collen D (1981) Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J. Biol. Chem. 256: 7035–7041.

    CAS  Google Scholar 

  • Rosengart TK, Johnson WV, Friesel R, Clark R and Maciag T (1988) Heparin protects heparin-binding growth factor-I from proteolytic inactivationin vitro. Biochem. Biophys. Res. Commun. 152: 441–448.

    Article  Google Scholar 

  • Rubin SH, Chan AM-L, Bottaro DP, Burgess WH, Taylor WG, Cech AC, Hirschfield DW, Wong J, Miki T, Finch PW and Aaronson SA (1991) A broad-spectrum human lung fibroblast-derived mitogen as a variant of hepatocyte growth factor. Proc. Natl. Acad. Sci. USA 88: 415–419.

    Article  CAS  Google Scholar 

  • Sano E, Kamata K, Okano K, Yamazaki S, Iizuka M and Kobayashi M (1988) Structural characterization of recombinant human interferon-gammas derived from two different mammalian cells. Microbiol. Immunol. 32: 499–510.

    CAS  Google Scholar 

  • Schreiber AB, Kenny J, Kowalski WJ, Friesel R, Mehlman T and Maciag T (1985) Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition. Proc. Natl. Acad. Sci. USA 82: 6138–6142.

    Article  CAS  Google Scholar 

  • Shimomura T, Honda T, Oouchi C, Kondo J and Nagaike K (1991) Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture. Cytotechnology 6: 1–11.

    Article  CAS  Google Scholar 

  • Sommer A and Rifkin DB (1989) Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan. J. Cell Physiol. 138: 215–220.

    Article  CAS  Google Scholar 

  • 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 

  • Travis J and Salvesen GS (1983) Human plasma proteinase inhibitors. Ann. Rev. Biochem. 52: 655–709.

    Article  CAS  Google Scholar 

  • Umezawa H (1976) In: Lorand L (ed.) Methods in Enzymology. Academic Press, New York, 45: 678.

    Google Scholar 

  • Wallen P, Bergsdorf N and Ranby M (1982) Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin. Biochim. Biophys. Acta 719: 318–328.

    CAS  Google Scholar 

  • Yoshiyama Y, Arakaki N, Naka D, takahashi K, Hirono S, Kondo J, Nakayama H, Gohda E, Kitamura N, Tsubouchi H, Ishii T, Hishida T and Daikuhara Y (1991) Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 175: 660–667.

    Article  CAS  Google Scholar 

  • Zarnegar R and Michalopoulos G (1989) Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res. 49: 3314–3320.

    CAS  Google Scholar 

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Shimomura, T., Ochiai, M., Kondo, J. et al. A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to two chain form in serum-free culture. Cytotechnology 8, 219–229 (1992). https://doi.org/10.1007/BF02522039

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  • DOI: https://doi.org/10.1007/BF02522039

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