Preparation, Characterization and Refolding in Vitro of a Recombinant Human Cyclophilin A Mutant: Effect of a Single Pro/Ser Substitution on Cyclophilin A Structure and Properties

摘要

A recombinant cyclophilin A (CypA) mutant, which carries a serine instead of proline at sequence 16, was prepared for structural and functional assessment for human CypA. Soluble expression of the recombinant CypA mutant in E. coli was obtained under 30 °C, 180 rpm culture condition after being induced by IPTG. Ion exchange chromatography was used to purify the CypA mutant in a single step, and a high activity recovery of target protein with a high purity was achieved. Peptide fragments produced by trypsin proteolysis were applied to MALDI-TOF-MS, and searching results from the NCBI protein databank confirmed the protein attribution as well as the mutation sequence. Peptidyl-prolyl cis-trans isomerase activity was assayed for the CypA mutant using tetrapeptide substrate Sue-Ala-Ala-Pro-Phe-p-nitroanilide, and the calculated k_(cat)/ K_m value was 1.5 x 10~6 M~(-1) s~(-1) at 10 °C, which was 10-fold lower than the-previously reported constant for wild-type CypA. An Eyring plot was also carried out. Inhibition by cyclosporine A demonstrated that the IC_(50) value was 26.5 nM. Meanwhile the expected enhancement of intrinsic tryptophan fluorescence was quenched by the mutation. The effect of CypA mutant on accelerating protein refolding in vitro was investigated in ribonuclease A refolding process, and it was found that 10% slow phase could be catalyzed by CypA. The protein was subject to urea and GdmCl denaturation, where both activity and fluorescence served as structural probes. Activity recovery indicated this CypA mutant was extremely sensitive to GdmCl and the susceptibility to urea was increased. Low pH could also destabilize CypA. Furthermore the refolding of this CypA mutant itself was studied. Although the activity yield was nearly unchanged, the former proposed folding/assembly pathway might be altered. Fluorescence chart also demonstrated that the folding time was extended, and fast-folding and slow-folding analysis indicated the slow-folding rate constant presented a concentration dependence property denoting the autocatalysis of the foldase.