Expression of Cu, Zn-superoxide dismutase gene from Saccharomyces cerevisiae in Pichia pastoris and its resistance to oxidative stress

Authors: Li, Jian-Rong¹; Yu, Ping²

Source: Applied Biochemistry and Biotechnology, Volume 136, Number 1, February 2007 , pp. 127-139(13)

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Abstract:

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.

Keywords: Cu; Zn-superoxide dismutase gene; polymerase chain reaction; sequence analysis; similarity comparison; oxidative stress

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s12010-007-0022-7

Affiliations: 1: College of Food Science, Biotechnology and Environmental Engineering, Zhejiang Gongshang University, 310035, Hangzhou, People's Republic of China, 2: College of Food Science, Biotechnology and Environmental Engineering, Zhejiang Gongshang University, 310035, Hangzhou, People's Republic of China, Email: yup9202@yahoo.com.cn

Publication date: 2007-02-01