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ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(4): 234–240 CN 31-1940/Q

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Expression of Monomeric Insulin Precursor in Pichia pastoris and Its Conversion into Monomeric B27 Lys Destripeptide Insulin by Tryptic Hydrolysis

Jin-Guo DING1,3, Jian FEI2, Da-Fu CUI1*, and You-Shang ZHANG1*

1Institute of Biochemistry and Cell Biology, and

2Model Organism Research Center, Shanghai Institutes for Biological Sciences,

the Chinese Academy of Sciences, Shanghai 200031, China;

3Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China

Abstract Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition, and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.

Key words monomeric B27 Lys destripeptide insulin; tryptic hydrolysis; Pichia pastoris

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Received: February 4, 2005 Accepted: March 2, 2005

*Corresponding authors:

Da-Fu CUI: Tel, 86-21-54921262; Fax, 86-21-54921011; E-mail, [email protected]

You-Shang ZHANG: Tel, 86-21-54921237; Fax, 86-21-54921011; E-mail, [email protected]