Secretory expression of a single-chain insulin precursor in yeast and its conversion into human insulin.
A synthetic single-chain porcine insulin precursor (PIP) gene and an alpha-mating factor leader sequence (alpha MFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/alpha MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cerevisiae transformed by pVT102-U/alpha MFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.
Zhang
Y
Y
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, China.
Hu
H
H
Cai
R
R
Feng
Y
Y
Zhu
S
S
He
Q
Q
Tang
Y
Y
Xu
M
M
Xu
Y
Y
Zhang
X
X
Liu
B
B
Liang
Z
Z
D001483:Base Sequence; D003001:Cloning, Molecular; D005813:Genes, Synthetic; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D007328:Insulin; D010455:Peptides; D011498:Protein Precursors; D000072235:Mating Factor