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  • Title: Studies on the binding of alpha-crystallin to recombinant prochymosins and chymosin.
    Author: Chitpinityol S, Goode D, Crabbe MJ.
    Journal: Mol Vis; 1998 Jan 16; 4():1. PubMed ID: 9485484.
    Abstract:
    PURPOSE: To further investigate the binding of alpha-crystallin to other proteins as part of its chaperone-like activity, we studied interactions of alpha-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 alpha-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. RESULTS: Refolding of 1.0 mg/ml of protein inclusion bodies diluted in phosphate buffer at 0.32 M urea in the presence of alpha-crystallin resulted in enhanced chymosin activity relative to the control without alpha-crystallin. When lower inclusion body concentrations were used, enzyme activity was not enhanced relative to the control. The mutant enzyme (PC+2) showed no conversion to the active form in the presence of alpha-crystallin. alpha-Crystallin formed a complex with refolded prochymosin, as well as with prochymosin during refolding, but not with active chymosin. Removal of the 43-residue propeptide resulted in loss of alpha-crystallin binding. The addition of two residues (Histidine-Glycine) to the prochymosin C-terminus resulted in precipitation of the mutant prochymosin-alpha-crystallin complex and loss of enzyme activity. CONCLUSIONS: Our experiments show that even under stringent refolding conditions, alpha-crystallin, which retains its gross oligomeric integrity, can bind to unfolded proteins in inclusion bodies and enhance the apparent yield of chymosin activity from high concentrations of inclusion bodies. alpha-Crystallin shows some specificity for binding to its target protein; this specificity may be based on steric considerations as well as residue-specific interactions.
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