Journal of Protein Chemistry

, Volume 11, Issue 3, pp 213–223

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

  • Rohinton Edalji
  • Tami J. Pilot-Matias
  • Steven D. Pratt
  • David A. Egan
  • Jean M. Severin
  • Earl G. Gubbins
  • Andrew M. Petros
  • Stephen W. Fesik
  • Neal S. Burres
  • Thomas F. Holzman
Article

DOI: 10.1007/BF01024859

Cite this article as:
Edalji, R., Pilot-Matias, T.J., Pratt, S.D. et al. J Protein Chem (1992) 11: 213. doi:10.1007/BF01024859

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.

Key words

FK-binding protein CKS-FKBP fusion protein limited proteolysis peptidyl prolyl isomerase 

Copyright information

© Plenum Publishing Corporation 1992

Authors and Affiliations

  • Rohinton Edalji
    • 1
  • Tami J. Pilot-Matias
    • 5
  • Steven D. Pratt
    • 5
  • David A. Egan
    • 1
  • Jean M. Severin
    • 1
  • Earl G. Gubbins
    • 2
  • Andrew M. Petros
    • 3
  • Stephen W. Fesik
    • 3
  • Neal S. Burres
    • 4
  • Thomas F. Holzman
    • 1
  1. 1.Protein Biochemistry, Structural Biology UnitAbbott LaboratoriesAbbott Park
  2. 2.Molecular BiologyAbbott LaboratoriesAbbott Park
  3. 3.NMR Research, Drug Design and DeliveryAbbott LaboratoriesAbbott Park
  4. 4.Anti-Infective Research, Pharmaceutical Products DivisionAbbott LaboratoriesAbbott Park
  5. 5.Corporate Molecular Biology, Abbott Diagnostics DivisionAbbott LaboratoriesAbbott Park
  6. 6.Protein Biochemistry, D-46Y, AP-10-1, Discovery ResearchAbbott LaboratoriesAbbott Park