title: Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.
abstract: In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.
authors.affiliation: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yang-Bin
authors.initials: YB
authors.lastname: Huang
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
authors.forename: Jiu-Ru
authors.initials: JR
authors.lastname: Sun
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Tao
authors.initials: T
authors.lastname: Feng
authors.forename: Jian
authors.initials: J
authors.lastname: Fei
authors.forename: Da-Fu
authors.initials: DF
authors.lastname: Cui
authors.forename: Qi-Chang
authors.initials: QC
authors.lastname: Xia
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.forename: You-Shang
authors.initials: YS
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002096:C-Peptide / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002096:C-Peptide; D011498:Protein Precursors