Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product.
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.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Y
Y
Wang
Z H
ZH
Liang
Y S
YS
Zhang
S Y
SY
Yao
Y G
YG
Xu
Y H
YH
Tang
S Q
SQ
Zhu
D F
DF
Cui
Y M
YM
Feng
D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014170:Transformation, Genetic; D015032:Zinc / Q000378:metabolism
D007328:Insulin; D011498:Protein Precursors; D011994:Recombinant Proteins; D011972:Receptor, Insulin; D015032:Zinc