title: Analysis of the disulfide bonding pattern between domain sequences of recombinant prochymosin solubilized from inclusion bodies.
abstract: Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins.
authors.affiliation: Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, Beijing.
authors.forename: C
authors.initials: C
authors.lastname: Wei
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Yang
mesh_terms: D012085:Chymosin / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility
chemical_list: D004220:Disulfides; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin