The cDNA encoding a human cytosolic 40‐kDa cyclophilin (CyP‐40) has been inserted into a modified pGEX‐3X expression vector and expressed in Escherichia coli to yield recombinant CyP‐40 at levels up to 4 mg/1 medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP‐40 showed peptidyl‐prolyl cis‐trans isomerase activity (kcat/Km= 1.12×106 M−1· s−1) comparable to that of bovine brain CyP‐40. The weak affinity of CyP‐40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site‐directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20‐fold greater affinity for cyclosporin A (Kdapp 11.5±2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl‐Ala‐Ala‐Pro‐Phe 4‐nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP‐40 and had an activity similar to that of CyP‐18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin‐A‐binding domain that is similar to that of CyP‐18.
|Original language||English (US)|
|Number of pages||6|
|Journal||European Journal of Biochemistry|
|State||Published - Apr 1995|
- cyclosporin A