Role of the amino-terminal amino acid sequences determining the in vitro refolding process of prochymosin polypeptide.

Prochymosin, a zymogen of an aspartic proteinase chymosin, is produced as inclusion bodies in the recombinant Escherichia coli cells. Solubilization of the inclusion bodies with 8 M urea followed by dialysis at pH 10.5 achieves correctly refolded prochymosin to some extent, which is then activated by self-processing at acidic pHs. Analyses of the alkaline dialysates by anion exchange chromatography revealed broad distribution of prochymosin polypeptides with different conformations. Stepwise dialysis with a slower decreasing rate of urea resulted in marked improvement of the yield of correctly refolded molecules. A hybrid prochymosin (CR601) possessing a short NH2-terminal replacement with the trp-leader peptide was not refolded into the correct conformation by one-step dialysis, but it was by stepwise dialysis. Replacement of Lys at the NH2-terminal second position of CR601 with Asp or Glu caused marked enhancement of correct refolding. These findings suggest that the amino acid sequence in the NH2-terminal region of prochymosin plays a crucial role in determining the whole refolding process of the polypeptide.