Studies on the Binding of α−Crystallin to Recombinant Prochymosins and Chymosin

Purpose: To further investigate the binding of α−crystallin to other proteins as part of its chaperone-like activity, we studied interactions of α−crystallin with recombinant calf prochymosins and chymosin. Methods: Recombinant calf prochymosin B and one C-terminal mutant (PC+2, with two additional residues, Histidine-Glycine) were expressed as inclusion bodies in E. coli. Native and mutant proteins were denatured in 8 M urea before being refolded by dilution slowly in phosphate buffer, pH 10.7, in the presence and absence of α−crystallin at different concentration ratios. After dialysis, the folded proteins were converted to the active chymosin by acidification. The resulting enzyme activities at standard protein concentrations were determined by a microtitre milk-clotting assay.