title: Studies on the binding of alpha-crystallin to recombinant prochymosins and chymosin.
abstract: OBJECTIVE
  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.
authors.affiliation: Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, The University of Reading, UK.
authors.forename: S
authors.initials: S
authors.lastname: Chitpinityol
authors.forename: D
authors.initials: D
authors.lastname: Goode
authors.forename: M J
authors.initials: MJ
authors.lastname: Crabbe
mesh_terms: D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000378:metabolism*; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D002479:Inclusion Bodies / Q000201:enzymology; D008892:Milk / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D003459:Crystallins; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin