Advertisement

Journal of Protein Chemistry

, Volume 12, Issue 3, pp 323–327 | Cite as

Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies

  • John O. Hui
  • Alfredo G. Tomasselli
  • Ilene M. Reardon
  • June M. Lull
  • David P. Brunner
  • Che-Shen C. Tomich
  • Robert L. Heinrikson
Article

Abstract

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofMr ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

Key words

HIV-1 protease gel-filtration Superdex 75 FPLC column reversed-phase HPLC 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ashorn, P., McQuade, T. J., Thaisrivongs, S., Tomasselli, A. G., Tarpley, W. G., and Moss, B. (1990).Proc. Natl. Acad. Sci. USA 87, 7472–7476.Google Scholar
  2. Copeland, T. D., and Oroszlan, S. (1988).Gene Anal. Tech. 5, 109–115.Google Scholar
  3. Ido, E., Han, H-P., Kezdy, F. J., and Tang, J. (1991).J. Biol. Chem. 266, 24,359–24,366.Google Scholar
  4. Kohl, N. E., Emini, E. A., Schleif, W. A., Davis, L. J., Heimbach, J. C., Dixon, R. A. F., Scolnick, E. M., and Sigal, I. S. (1988).Proc. Natl. Sci. USA 85, 4686–4690.Google Scholar
  5. Laemmli, U. K. (1970).Nature 227, 680–684.Google Scholar
  6. Leatherbarrow, R. J. (1988).Enzfitter.Google Scholar
  7. Nutt, R. F., Brady, S. F., Darke, P. L., Ciccarone, T. M., Colton, C. D., Nutt, E. M., Rodkey, J. A., Bennett, C. D., Waxman, L. H., Sigal, I. S., Anderson, P. S., and Veber, D. F. (1989).Proc. Natl. Acad. Sci. USA 85, 7129–7133.Google Scholar
  8. Pichuantes, S., Babé, L. M., Barr, P. J., and Craik, C. S. (1989).Proteins: Structure, Function and Genetics 6, 324–337.Google Scholar
  9. Schneider, J., and Kent, S. B. H. (1988).Cell 54, 363–368.Google Scholar
  10. Strickler, J. E., Gorniak, J., Dayton, B., Meek, T., Moore, M., Mazaard, V., Malinowski, J., and Debouck, C. (1989).Proteins: Structure, Function and Genetics 6, 139–154.Google Scholar
  11. Thaisrivongs, S., Tomasselli, A. G., Moon, J. B., Hui, J., McQuade, T. J., Turner, S. R., Strobach, J. W., Howe, W. J., Tarpley, W. G., and Heinrikson, R. L. (1991).J. Med. Chem. 34, 2344–2356.Google Scholar
  12. Thanki, N., Mohana Rao, J. K., Foundling, S. I., Howe, W. J., Moon, J. B., Hui, J. O., Tomasselli, A. G., Heinrikson, R. L., Thaisrivongs, S., and Wlodawer, A. (1992).Protein Science 1, 1061–1072.Google Scholar
  13. Tomasselli, A. G., Olsen, M. K., Hui, J. O., Staples, D. J., Sawyer, T. K., Heinrikson, R. L., and Tomich, C.-S. (1990a).Biochemistry 29, 264–269.Google Scholar
  14. Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., Bannow, C., Reardon, I. M., Howe, W. J., DeCamp, D. L., Craik, C. S., and Heinrikson, R. L. (1990b).J. Biol. Chem. 265, 14,675–14,683.Google Scholar
  15. Tomasselli, A. G., Howe, W. J., Sawyer, T. K., Wlodawer, A., and Heinrikson, R. L. (1991).Chemistry Today—Chimica Oggi (Milan)9, 6–27.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • John O. Hui
    • 1
  • Alfredo G. Tomasselli
    • 1
  • Ilene M. Reardon
    • 1
  • June M. Lull
    • 1
  • David P. Brunner
    • 1
  • Che-Shen C. Tomich
    • 1
  • Robert L. Heinrikson
    • 1
  1. 1.The Upjohn CompanyKalamazoo

Personalised recommendations