Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris |
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Authors: | Huang Yang-Bin Li Jiang Gao Xin Sun Jiu-Ru Lu Yi Feng Tao Fei Jian Cui Da-Fu Xia Qi-Chang Ren Jun Zhang You-Shang |
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Institution: | Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. |
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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. Key words proinsulin; C-peptide; Pichia pastoris |
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Keywords: | proinsulin C-peptide Pichia pastoris |
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