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Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product
Author(s) -
Wang Yan,
Liang ZhenHe,
Zhang YouShang,
Yao ShiYin,
Xu YingGao,
Tang YueHua,
Zhu ShangQuan,
Cui DaFu,
Feng YouMin
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20010405)73:1<74::aid-bit1038>3.0.co;2-v
Subject(s) - pichia pastoris , heterologous , heterologous expression , recombinant dna , yeast , biochemistry , pichia , fermentation , biology , human insulin , insulin receptor , insulin , chemistry , microbiology and biotechnology , gene , insulin resistance
The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot‐blotting method. In high‐density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two‐step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 74–79, 2001.