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.
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Agarwal, R. P., Threatte, G. A., and McPherson, R. A. (1987).Clin. Chem. 33, 481–485.
Bachinger, H. P. (1987).J. Biol. Chem. 262, 17,144–17,148.
Bolling, T. J., and Mandecki, W. (1990).Biotechniques 8, 488–490.
Clore, G. M., Kay, L. E., Bax, A., and Gronenborn, A. M. (1991).Biochemistry 30, 12–18.
Fesik, S. W., and Zuiderweg, E. R. P. (1988).J. Magn. Res. 78, 588–593.
Fesik, S. W., and Zuiderweg, E. R. P. (1988).Q. Rev. Biophys. 23, 97–131.
Fischer, G., Bang, H., and Mech, C. (1984).Biomed. Biochim. Acta 43, 1101–1111.
Fischer, G., and Bang, H. (1985).Bioch. Biophys. Acta 828, 39–42.
Gill, S. C., and von Hippel, P. H. (1989).Anal. Biochem. 182, 319–326.
Handschumacher, R. E., Harding, M. W., Rice, J., Drugge, R. J., and Speicher, D. W. (1984).Science 226, 544–547.
Harding, M. W., Handschumacher, R. E., and Speicher, D. W. (1986).J. Biol. Chem. 261, 8547–8555.
Harding, M. W., Galat, A., Vehling, D. E., and Schreiber, S. L. (1989).Nature 341, 758–760.
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.
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.
Holzman, T. F., Fesik, S. W., Park, C. H., and Kofron, J. (1991b). InEnzymes in Biotechnology, Plenum, New York, (in press).
Kay, L. E., Ikura, M., Tschudin, R., and Bax, A. (1990).J. Magn. Reson. 89, 496–514.
Kay, L. E., Clore, G. M., Bax, A., and Gronenborn, A. M. (1990).Science 249, 411–414.
Maki, N., Sekiguchi, F., Nishimaki, J., Miwa, K., Hayano, T., Takahashi, N., and Suzuki, M. (1990).Proc. Natl. Acad. Sci., USA 87, 5440–5443.
Marion, D., Kay, L. E., Sparks, S. W., Torchia, D. A., and Bax, A. (1989).J. Am. Chem. Soc. 111, 1515–1517.
Lane, W. S., Galat, A., Harding, M. W., and Schreiber, S. L. (1991)J. Prot. Chem. 10, 151–160.
Liu, J., Albers, M. W., Chen, C., Schreiber, S. L., and Walsh, C. T. (1990).Proc. Natl. Acad. Sci. USA 87, 2304–2308.
Michnick, S. W., Rosen, M. K., Wandless, T. J., Karplus, M., and Schreiber, S. L. (1991).Science 252, 836–839.
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.
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.
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.
Standaert, R. F., Galat, A., Verdine, G. L., and Schreiber, S. L. (1990).Nature 346, 671–674.
Tropschug, M., Wachter, E., Majer, S., Schonbrunner, R., and Schmidt, F. X. (1990).Nature 346, 674–677.
Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1991).Science 252, 839–842.
Wuthrich, K. (1986). InNMR of Proteins and Nucleic Acids, John Wiley & Sons, New York.
Zuiderweg, E. R. P., Petros, A. M., Fesik, S. W., and Olejniczak, E. T. (1991).J. Am. Chem. Soc. 113, 370–372.
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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
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DOI: https://doi.org/10.1007/BF01024859