Preparation, Characterization Of CypA And The Preliminary Application In Protein Refolding In Vitro

Cyclophilin A (CypA) is one of peptidyl-prolyl cis/trans isomerases (PPIase). CypA catalyzes the cis/trans isomerization of Xaa-Pro peptide bonds, and thus promotes protein folding in vitro and in vivo in which proline isomerization is the rate-limiting step. The in vitro study of CypA assisted protein refolding is quite significant for both theoretical and industrial consideration: it can help us understand the folding mechanism of the de novo protein folding, and also it will contribute to industrial renaturation of proteins expressed as inclusion bodies.Firstly, CypA coding plasmid was successfully transformed, and culture conditions of the recombinant E. coli were investigated. Intracellular soluble expression of recombinant CypA was achieved by controlling temperature and rotaing speed. Then, the culture medium and other operative conditions were optimized. Finally, we used 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 10 g/L mannitol for the medium, and pH was adjusted to 7.0. Amp was added to 20 mg/L. Loading ratio was 200 mL/500 mL, and inoculum was 10%. Inducer IPTG was added to 1 mM when OD₆₀₀ achieved 1.3. After inducement, the culture was shifted to 30℃,180 rpm. E. coli was harvested after 9 h further culture. Soluble target protein expression was 66.7 mg per liter fermentation broth. Then, ion-exchange chromatography was used to purify CypA from supernatant of cells lysate. The protein recovery was 35.5%, activity recovery was as high as 82.4%, and 54.9 mg high purity grade (SDS-PAGE pure) CypA was obtained per liter fermentation broth.Secondly, Pro16/Ser16 substitution was highlighted in the characterization of CypA structure and functions, including peptide mass fingerprint, PPIase activity assay, cyclosporine A (CsA) inhibition assay, denaturation curves and CypA refolding. K_cat/K_m constant of CypA catalysis toward tetrapeptide was found to be 1.5×10⁶ M^-1S^-1, which was 10-fold lower than wild-type CypA. IC₅₀ was found to be 26.5 nM and consistent with wild-type. Erying plots were carried to study thermodynamic properties of proline isomerization. CypA showed susceptibility to GdmCl, and it was less stable under urea incubation. CypA was found to occupy auto-catalysis characteristics in its refolding process. After a series of assays, Pro16/Ser16 substitution was concluded to play an important role in CypA structure and functions.Finally, some attempts were made to evaluate functions of CypA in protein refolding in vitro. The two substrates used were RNase A and G-CSF, respectively. CypA can accelerate the slow-folding phase of RNase A when a kinetics analysis was carried out. The function of CypA in G-CSF refolding was more complicated. RP-HPLC results showed that CypA could indeed promote the refolding of G-CSF in the process, but the mechanism was not clear enough. One possible explanation was: firsly, PPIase activity of CypA helped quick formation of correct G-CSF isomers so that disulfide bridges in these molecules were easier to be linked; in further folding process, CypA may also exert its chaperone activity.