Construction of prokaryotic expression vector, expression and purification of ginseng Cu/Zn superoxide dismutase.
- Author:
Hong-Mei LIN
;
Ze-Yu WANG
;
Yue SHAO
;
Xiao-Ye QIN
;
Shi-Chao LIU
;
Xin ZHANG
;
Li-Min YANG
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
Gene Expression;
Genetic Engineering;
methods;
Genetic Vectors;
genetics;
Panax;
enzymology;
genetics;
Sequence Analysis;
Superoxide Dismutase;
genetics;
isolation & purification;
metabolism
- From:
China Journal of Chinese Materia Medica
2013;38(23):4052-4055
- CountryChina
- Language:Chinese
-
Abstract:
The total RNA was extracted from ginseng leaves of Panax ginseng. The Cu/Zn-SOD gene was amplified via RT-PCR and the pET-28(a)-Cu/Zn-SOD expression vector was constructed. The pET-28 (a)-Cu/Zn-SOD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and was induced by IPTG in order to select optimal induction of expression conditions. The target protein was purified by the nickel ions (Ni ) affinity chromatography and the target protein enzyme activity was determinated by the xanthine oxidase method. The similarity of the Cu/Zn-SOD gene sequences and the Cu/Zn-SOD gene sequences of Korean ginseng in NCBI was 99. 00%. The target protein expression level was about 44.42%, and the molecular weight was 16.30 kDa after the pET-28(a)-Cu/Zn-SOD recombinants were induced by IPTG. The purified Cu/Zn-SOD protease activity reached 10,596.69 U x mg(-1). The P. ginseng pET-28(a)-Cu/Zn-SOD prokaryotic expression vector was built by the method of molecular biology, which provided the foundation for studying the Cu/Zn-SOD biology function.
