Secretory Expression of
Human Insulin in Methylotrophic Yeast Pichia pastoris
WANG Yan, LIANG Zhen-He, ZHANG You-Shang, CUI Da-Fu1
and FENG You-Min*
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry；
1Shanghai Institute of Biochemistry, the Chinese Academy of
Sciences, Shanghai 200031, China )
Abstract The porcine insulin precursor
(PIP) gene and its derivative form sp-PIP gene, which had a
nona-peptide (called spacer peptide, sp) added at the 5′ terminus of PIP
gene, were inserted into the plasmid pPIC9 of Pichia pastoris to
obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP,
respectively. P.pastoris GS115 was transformed by pPIC9/PIP
or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were
selected by dot-blotting. The expression levels of PIP and sp-PIP were 10 mg/L
and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer
peptide could increase the expression. The expression level of PIP (sp-PIP)
in P.pastoris was higher than that of PIP in S.cerevisiae and
K.lactis reported in this laboratory. The expression level of sp-PIP
was 250 mg/L in 10 L fermentor. Recombinant human insulin was obtained by means
of transpeptidation of PIP or sp-PIP. The receptor binding capacity is
identical with that of porcine insulin. In vivo biological activity of
the recombinant human insulin is 27 IU/mg.
Key Words recombinant human insulin； porcine insulin precursor
(PIP)； Pichia pastoris； expression system
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