Human Proinsulin C-peptide from a Precursor Overexpressed in Pichia pastoris  

作　　者：Yang-Bin HUANG Jiang LI Xin GAO Jiu-Ru SUN Yi LU Tao FENG Jian FEI Da-Fu CUI Qi-Chang XIA Jun REN You-Shang ZHANG 

机构地区：[1]Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China [2]Shanghai Newsummit Biopharma Company, Shanghai 200233, China [3]Zhongshan Hospital Fudan University, Shanghai 200031, China [4]Shanghai Yizhong Biotechnology Company, Shanghai 201203, China [5]Shanghai Center of Research and Development of New Drugs, Shanghai 201203, China

出　　处：《Acta Biochimica et Biophysica Sinica》2006年第8期586-592,共7页生物化学与生物物理学报（英文版）

摘　　要：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 pastorisIn 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

关 键 词：PROINSULIN C-PEPTIDE Pichia pastoris 

分 类 号：Q5[生物学—生物化学]