Skip to main content
SpringerLink
Log in

Effector-assisted refolding of recombinant tissue-plasminogen activator produced in escherichia coli

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Recombinant tissue-plasminogen activator (r-tPA), expressed inEscherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained.

Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatograph (CPC), in scaling up the reactivation process or the purification step was also evaluated.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Marston, A. O. (1986),Biochem. J. 240, 1.

    CAS  Google Scholar 

  2. Sharma, S. K. (1986),Sep. Sci. Tech. 21(8), 710.

    Article  Google Scholar 

  3. Wilkox, G. and Studnicka, G. M. (1988),Biotech. Appl. Biochem. 10, 500.

    Google Scholar 

  4. Schein, C. H. (1989),Biotechnology 7, 1141.

    CAS  Google Scholar 

  5. Burton, S. J., Wood, P. C., and Quirk, A. V. (1989),Eur. f. Biochem. 179, 379.

    Article  CAS  Google Scholar 

  6. Latta, M., Knapp, M., Sarmientos, P., Brefort, G., Becquart, J., Guerrier, L., Jung, G., and Mayaux, J.-F. (1987),Biotechnology 5, 1309.

    Article  CAS  Google Scholar 

  7. Bachmann F. (1987), Thrombosis and Haemostasis, Verstraete, M., Vermylen, H. R., Lijnen, H. R., and Arnout, J., eds., pp. 227–265. International Society on Thrombosis and Haemostasis and Leuven University Press, Leuven, Belgium.

    Google Scholar 

  8. Banyai, L., Varadi, A., and Patthy, L. (1983),FEBS Lett. 163, 37.

    Article  CAS  Google Scholar 

  9. Caillaud, F., Latta, M., Mayaux, J. F., and Sarmientos, P. (1987), Eur. Pat. No. 87251699.

  10. Sarmientos, P., Duchesne, M., Denefle, P., Boiziau, J., Fromage, N., Delporte, N., Parker, F., Lelievre, Y., Mayaux, J. F., and Cartwright, T. (1989),Biotechnology 7, 495.

    Article  CAS  Google Scholar 

  11. Rudolph, R., Opitz, U., Hesse, F., Reisland, R., Fischer, S., and Mattes, R. (1987), German Pat. No. DE3611817 of Boehringer Mannheim GmbH.

  12. Albertsson, P.-A. (1986),Partition of Cell Particles and Macromolecules, 3rd Ed., Wiley & Sons, New York.

    Google Scholar 

  13. Walter, H., Brooks, D. E., Fisher, D., eds. (1985),Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology, Academic, New York.

    Google Scholar 

  14. Beauchamp, C. O., Gonias, S. L., Menapace, D. P., and Pizzo, S. V. (1983),Anal. Biochem. 131, 25.

    Article  CAS  Google Scholar 

  15. Connors, R. W., Sweet, R. W., Noveral, J., Trill, J. T., and Reff, M. (1988),DNA 7, 651.

    Article  CAS  Google Scholar 

  16. Shatzman, A. R., Gross, M. S., and Rosenberg, M. (1990),Current Protocols in Mol. Bio. Suppl.11, 16.3.1–16.3.11.

  17. Devore, S., Shatzman, A. R., Robbins, K., Rosenberg, M., and Aaronson, S. (1984),Cell 6, 43.

    Article  Google Scholar 

  18. Bradford, M. (1976),Anal. Biochem. 72, 248.

    Article  CAS  Google Scholar 

  19. Laemmli, U. K. (1970),Nature 227, 680.

    Article  CAS  Google Scholar 

  20. Conners, R. W., Sweet, R. W., Noveral, J. P., Pfarr, D. S., Trill, J. J., Shebuski, R. J., Berkowitz, B. A., Williams, D., Franklin, S., and Reff, M. E. (1988),DNA 7, 651.

    Google Scholar 

  21. Gaffney, P. J. and Curtis, A. D. (1985),Thromb. Haemostas. 53, 134.

    CAS  Google Scholar 

  22. Berthod, A. and Armstrong, D. W. (1988),J. Liq. Chrom. 11(3), 547.

    Article  CAS  Google Scholar 

  23. Rijken, D. C. and Collen, D. (1981),J. Biol. Chem. 256, 7035.

    CAS  Google Scholar 

  24. Pace, C. N. (1990),TIBS 15(1), 14.

    CAS  Google Scholar 

  25. Mitraki, A. and King, J. (1989),Biotechnology 7, 690.

    Article  CAS  Google Scholar 

  26. Leutzinger, Y. and Beychok, S. (1981),Proc. Natl. Acad. Sci. USA 78(2), 780.

    Article  CAS  Google Scholar 

  27. Teipel, J. W. and Koshland, D. E. (1971),Biochemistry 10(5), 792.

    Article  CAS  Google Scholar 

  28. Khan, I. A. and Nishimura, J. S. (1988),J. Biol. Chem. 263(5), 2152.

    CAS  Google Scholar 

  29. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennet, W. F., Yelverton, E., Seeburg, P. H., Heyneker, H. L., and Goeddel, D. V. (1983),Nature 301, 214.

    Article  CAS  Google Scholar 

  30. Harris, T. J. R., Patel, T., Marston, F. A. O., Little, S., Emtage, J. S., Opdenakker, G., Volckaert, G., Rombauts, W., Billiau, A., and De Somer, P. (1986),Mol. Biol. Med. 3, 279.

    CAS  Google Scholar 

  31. Griffiths, J. B. and Electricwale, A. (1987),Adv. Biochem. Eng. 34, 147.

    CAS  Google Scholar 

  32. Rhodes, M. and Birch, J. (1988),Biotechnology 6, 518.

    Article  CAS  Google Scholar 

  33. Cleland, J. L. and Wang, D. I. C. (1990),Biotechnology 8, 1274.

    Article  CAS  Google Scholar 

  34. Marston, F. A., Angal, S., Lowe, P. A., Chan, M., and Hill, C. R. (1988),Biochem. Soc. Trans. 16(2), 112.

    CAS  Google Scholar 

  35. Kacian, D. L. and Harvey, R. C. (1985),Arch. Biochem. Biophys. 236(1), 354.

    Article  CAS  Google Scholar 

  36. Husain, S. S., Lipinski, B., and Gurewich, V. (1981),Proc. Natl. Acad. Sci. USA 78(7), 4265.

    Article  CAS  Google Scholar 

  37. Reagan, M. E., Robb, M., Bornstein, I., and Niday, E. G. (1985),Thromb. Res. 40, 1.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Protein Biochemistry, Smith Kline Beecham Pharmaceuticals, 709 Swedeland Rd., 19406, King of Prussia, PA

    H. Grcinfeld, A. Patel & A. H. Nishikawa

  2. Departments of Gene Expression Sciences, Smith Kline Beecham Pharmaceuticals, 709 Swedeland Rd., 19406, King of Prussia, PA

    A. Shatzman

Authors
  1. H. Grcinfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Shatzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. H. Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grcinfeld, H., Patel, A., Shatzman, A. et al. Effector-assisted refolding of recombinant tissue-plasminogen activator produced in escherichia coli. Appl Biochem Biotechnol 33, 117–138 (1992). https://doi.org/10.1007/BF02950781

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02950781

Index Entries

Search

Navigation