Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.
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.
Huang
Yang-Bin
YB
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Li
Jiang
J
Gao
Xin
X
Sun
Jiu-Ru
JR
Lu
Yi
Y
Feng
Tao
T
Fei
Jian
J
Cui
Da-Fu
DF
Xia
Qi-Chang
QC
Ren
Jun
J
Zhang
You-Shang
YS
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
D002096:C-Peptide; D011498:Protein Precursors