A new approach to the production of the recombinant SOD protein by methylotrophic Pichia pastoris.
The gene for the copper, zinc-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 yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.
Yu
Ping
P
College of Food Science, Biotechnology and Environmental Engineering, Zhejiang Gongshang University, Hangzhou, 310035, People's Republic of China. yup9202@yahoo.com.cn
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D013482:Superoxide Dismutase