Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 1997 Sep;24(3):227–234. doi: 10.1023/A:1007988713571

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

Karen Hansen, Marianne Kjalke, Poul Baad Rasmussen, Leif Kongerslev, Mirella Ezban
PMCID: PMC3449617  PMID: 22358766

Abstract

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of 35S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII.

Keywords: CHO cells, multicatalytic proteinase complex, proteases, protease inhibitors, proteasome, pulse labelling, recombinant coagulation factor VIII, SDS-PAGE

Full Text

The Full Text of this article is available as a PDF (80.4 KB).

References

  1. Andersson L-O, Brown JE. Interaction of factor VIII — von Willebrand factor with phospholipid vesicles. Biochem. J. 1981;200:161–167. doi: 10.1042/bj2000161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ezban M, Chapman B, Petersen J, Hansen K, Rasmussen PB, Thomsen J, Kjalke M, Kongerslev L. Characterization of recombinant human two-chain factor VIII. Thromb. Hemostasis. 1993;69:1088. [Google Scholar]
  3. Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Preneta PE, Stephens PE, Thomsen S, Yarranton GT, et al. The development of a process for the production of HIV1 GP120 from recombinant cell lines. In: Spier RE, et al., editors. Production of Biologicals from animal cells in culture. Oxford: Butterworth-Heinemann; 1991. pp. 110–114. [Google Scholar]
  4. Gilbert GE, Drinkwater D. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine. Biochemistry. 1993;32:9577–9585. doi: 10.1021/bi00088a009. [DOI] [PubMed] [Google Scholar]
  5. Hames BD. Preparation and electrophoresis of polyacrylamide gels. In: Hames BD, Rickwood D., editors. Gel electrophoresis of proteins. A practical approach. Oxford: Oxford University Press; 1990. pp. 30–50. [Google Scholar]
  6. Hosoi S, Miyaji H, Satoh M, Kurimoto T, Mihara A, Fujiyoshi N, Itoh S, Sato S. Optimization of cell culture conditions for production of biologically active proteins. Cytotechnology. 1991;5:S17–34. doi: 10.1007/BF00573878. [DOI] [PubMed] [Google Scholar]
  7. Kjalke M, Heding A, Talbo G, Persson E, Thomsen J, Ezban M. Amino acid residues 721–729 are required for full factor VIII activity. Eur. J. Biochem. 1995;234:773–779. doi: 10.1111/j.1432-1033.1995.773_a.x. [DOI] [PubMed] [Google Scholar]
  8. Kobayashi N, Takenouchi T, Endo S, Munekata E. 1H-NMR study of the conformation of bacitracin A in aqueous solution. FEBS Lett. 1992;305:105–109. doi: 10.1016/0014-5793(92)80874-G. [DOI] [PubMed] [Google Scholar]
  9. Kratje RB, Lind W, Wagner R. Evaluation of the proteolytic potential of in vitro-cultivated hybridoma and recombinant mammalian cells. J. of Biotechnology. 1994;32:107–125. doi: 10.1016/0168-1656(94)90174-0. [DOI] [PubMed] [Google Scholar]
  10. Mäkinen KK. Inhibition by bacitracin of some hydrolytic enzymes. Int. J. Protein Res. 1972;4:21–28. doi: 10.1111/j.1399-3011.1972.tb03394.x. [DOI] [PubMed] [Google Scholar]
  11. McMullen BA, Fujikawa K, Davie EW, Hedner U, Ezban M. Location of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A) Protein Sci. 1995;4:740–746. doi: 10.1002/pro.5560040413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mizunaga T, Katakura Y, Miura T, Maruyama Y. Purification and charactirization of yeast protein disulfide isomerase. J. Biochem. 1990;108:846–851. doi: 10.1093/oxfordjournals.jbchem.a123291. [DOI] [PubMed] [Google Scholar]
  13. Orlowski M. The multicatalytic proteinase complex, a major extralysosomal proteolytic system. Biochemistry. 1990;29:10289–10297. doi: 10.1021/bi00497a001. [DOI] [PubMed] [Google Scholar]
  14. Rasmussen PB, Nordfang O, Hansen K, Kongerslev L, Ezban M. Recombinant two-chain factor VIII obtained in high amounts at decreased temperature. Thromb. Haemost. 1993;69:1086. [Google Scholar]
  15. Rivett AJ. Proteasomes: multicatalytic proteinase complexes. Biochem. J. 1993;291:1–10. doi: 10.1042/bj2910001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roth RA. Bacitracin: An inhibitor of the insulin degrading activity of glutathione-insulin transhydrogenase. Biochem. Biophys. Res. Commun. 1981;98:431–438. doi: 10.1016/0006-291X(81)90858-5. [DOI] [PubMed] [Google Scholar]
  17. Toscano WA, Storm DR. Bacitracin. Pharmac. Ther. 1982;16:199–210. doi: 10.1016/0163-7258(82)90054-7. [DOI] [PubMed] [Google Scholar]
  18. Urlaub G, Mitchell PJ, Kas E, Chasin LA, Funanage VL, Myoda TT, Hamlin J. Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions. Somatic Cell and Molecular Genetics. 1986;12:555–566. doi: 10.1007/BF01671941. [DOI] [PubMed] [Google Scholar]
  19. Varandani PT, Shroyer LA, Nafz MA. Sequential degradation of insulin by rat liver homogenates. Proc. Natl. Acad. Sci. USA. 1972;69:1681–1684. doi: 10.1073/pnas.69.7.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES