Skip to main content
Log in

High-level expression of recombinant human FK-binding protein from a fusion precursor

  • Published:
Journal of Protein Chemistry Aims and scope Submit manuscript

Abstract

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L inEscherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a ∼30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed inE. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.

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

Access this article

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

  • Agarwal, R. P., Threatte, G. A., and McPherson, R. A. (1987).Clin. Chem. 33, 481–485.

    Article  CAS  PubMed  Google Scholar 

  • Bachinger, H. P. (1987).J. Biol. Chem. 262, 17,144–17,148.

    Article  CAS  Google Scholar 

  • Bolling, T. J., and Mandecki, W. (1990).Biotechniques 8, 488–490.

    CAS  PubMed  Google Scholar 

  • Clore, G. M., Kay, L. E., Bax, A., and Gronenborn, A. M. (1991).Biochemistry 30, 12–18.

    Article  CAS  PubMed  Google Scholar 

  • Fesik, S. W., and Zuiderweg, E. R. P. (1988).J. Magn. Res. 78, 588–593.

    Google Scholar 

  • Fesik, S. W., and Zuiderweg, E. R. P. (1988).Q. Rev. Biophys. 23, 97–131.

    Article  Google Scholar 

  • Fischer, G., Bang, H., and Mech, C. (1984).Biomed. Biochim. Acta 43, 1101–1111.

    CAS  PubMed  Google Scholar 

  • Fischer, G., and Bang, H. (1985).Bioch. Biophys. Acta 828, 39–42.

    CAS  Google Scholar 

  • Gill, S. C., and von Hippel, P. H. (1989).Anal. Biochem. 182, 319–326.

    Article  CAS  PubMed  Google Scholar 

  • Handschumacher, R. E., Harding, M. W., Rice, J., Drugge, R. J., and Speicher, D. W. (1984).Science 226, 544–547.

    Article  CAS  PubMed  Google Scholar 

  • Harding, M. W., Handschumacher, R. E., and Speicher, D. W. (1986).J. Biol. Chem. 261, 8547–8555.

    Article  CAS  PubMed  Google Scholar 

  • Harding, M. W., Galat, A., Vehling, D. E., and Schreiber, S. L. (1989).Nature 341, 758–760.

    Article  CAS  PubMed  Google Scholar 

  • Holzman, T. F., Chung, C. C., Edalji, R., Egan, D. A., Gubbins, E. J., Ruetter, A., Yang, L., Pederson, T. M., Krafft, G. A., and Wang, G. T. (1990).J. Prot. Chem. 9, 663–672.

    Article  CAS  Google Scholar 

  • Holzman, T. F., Egan, D. A., Edalji, R., Simmer, R. S., Helfrich, R., Taylor, A., and Burres, N. S. (1991a).J. Biol. Chem. 266, 2474–2479.

    Article  CAS  PubMed  Google Scholar 

  • Holzman, T. F., Fesik, S. W., Park, C. H., and Kofron, J. (1991b). InEnzymes in Biotechnology, Plenum, New York, (in press).

    Google Scholar 

  • Kay, L. E., Ikura, M., Tschudin, R., and Bax, A. (1990).J. Magn. Reson. 89, 496–514.

    CAS  Google Scholar 

  • Kay, L. E., Clore, G. M., Bax, A., and Gronenborn, A. M. (1990).Science 249, 411–414.

    Article  CAS  PubMed  Google Scholar 

  • Maki, N., Sekiguchi, F., Nishimaki, J., Miwa, K., Hayano, T., Takahashi, N., and Suzuki, M. (1990).Proc. Natl. Acad. Sci., USA 87, 5440–5443.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marion, D., Kay, L. E., Sparks, S. W., Torchia, D. A., and Bax, A. (1989).J. Am. Chem. Soc. 111, 1515–1517.

    Article  CAS  Google Scholar 

  • Lane, W. S., Galat, A., Harding, M. W., and Schreiber, S. L. (1991)J. Prot. Chem. 10, 151–160.

    Article  CAS  Google Scholar 

  • Liu, J., Albers, M. W., Chen, C., Schreiber, S. L., and Walsh, C. T. (1990).Proc. Natl. Acad. Sci. USA 87, 2304–2308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Michnick, S. W., Rosen, M. K., Wandless, T. J., Karplus, M., and Schreiber, S. L. (1991).Science 252, 836–839.

    Article  CAS  PubMed  Google Scholar 

  • Obukowicz, M. G., Gustafson, M. E., Junger, K. D., Leimgruber, R. M., Wittwer, A. J., Wun, T. C., Warren, T. G., Bishop, B. F., Mathis, K. J., McPherson, D. T., Siegel, N. R., Jennings, M. G., Brightwell, B. B., Diazcollier, J. A., Bell, L. D., Craik, C. S., and Tacon, W. C. (1990).Biochemistry 29, 9737–9745.

    Article  CAS  PubMed  Google Scholar 

  • Petros, A. M., Gampe, R. T., Gemmecker, G., Neri, P., Holzman, T. F., Edalji, R., Hochlowski, J., Jackson, M., McAlpine, J., Luly, J. R., Pilot-Matias, T., Pratt, S., and Fesik, S. W. (1991).J. Med. Chem. (in press).

  • Siekerka, J. J., Hung, S. H. Y., Poe, M., Lin, C. S., and Sigal, N. H. (1989).Nature 341, 755–757.

    Article  Google Scholar 

  • Siekerka, J. J., Wiederrecht, G., Greulich, H., Boulton, D., Hung, S. H. Y., Cryan, J., Hodges, N. J., and Sigal, N. H. (1990).J. Biol. Chem. 265, 21,011–21,015.

    Article  Google Scholar 

  • Standaert, R. F., Galat, A., Verdine, G. L., and Schreiber, S. L. (1990).Nature 346, 671–674.

    Article  CAS  PubMed  Google Scholar 

  • Tropschug, M., Wachter, E., Majer, S., Schonbrunner, R., and Schmidt, F. X. (1990).Nature 346, 674–677.

    Article  CAS  PubMed  Google Scholar 

  • Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1991).Science 252, 839–842.

    Article  PubMed  Google Scholar 

  • Wuthrich, K. (1986). InNMR of Proteins and Nucleic Acids, John Wiley & Sons, New York.

    Book  Google Scholar 

  • Zuiderweg, E. R. P., Petros, A. M., Fesik, S. W., and Olejniczak, E. T. (1991).J. Am. Chem. Soc. 113, 370–372.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Edalji, R., Pilot-Matias, T.J., Pratt, S.D. et al. High-level expression of recombinant human FK-binding protein from a fusion precursor. J Protein Chem 11, 213–223 (1992). https://doi.org/10.1007/BF01024859

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01024859

Key words

Navigation