title: Role of the amino-terminal amino acid sequences determining the in vitro refolding process of prochymosin polypeptide.
abstract: 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.
authors.affiliation: Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yonezawa
authors.forename: J
authors.initials: J
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Horinouchi
authors.forename: T
authors.initials: T
authors.lastname: Beppu
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D012085:Chymosin / Q000737:chemistry*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D003956:Dialysis; D004792:Enzyme Precursors / Q000737:chemistry*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding
chemical_list: D000596:Amino Acids; D004269:DNA, Bacterial; D004792:Enzyme Precursors; C032443:prorennin; D012085:Chymosin