title: Acidic residues on the N-terminus of proinsulin C-Peptide are important for the folding of insulin precursor.
abstract: To investigate the role of C-peptide in the folding of insulin precursor, a series of C-peptide mutant proinsulin genes were constructed, overexpressed in Escherichia coli and the proteins purified. Correct disulfide linkages of these proteins were confirmed by both tryptic peptide mapping and insulin receptor binding analyses. In vitro refolding experiments were performed with the purified proteins and showed that mutations on the glycine-rich middle segment of C-peptide, GGGPGAG, and deletion of the C-terminal pentapeptide, EGSLQ, as well as mutations on the two pairs of dibasic residues at the two ends of C-peptide did not significantly affect the refolding yields. However, both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N-terminus of the C-peptide resulted in serious aggregation during refolding. The results indicate that the highly conserved acidic N-terminal part of C-peptide is very important for insulin precursor folding, and that C-peptide may have some intramolecular chaperone-like function in the folding of insulin precursor.
authors.affiliation: National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, People's Republic of China.
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Chen
authors.forename: Xing-Wen
authors.initials: XW
authors.lastname: Yang
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002096:C-Peptide / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D002096:C-Peptide; D004220:Disulfides; D011994:Recombinant Proteins; D011384:Proinsulin