Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli.
- Author:
Yu-Zhong LIU
;
Ye SHEN
;
Qi-Xian RONG
;
Wen-Yan WU
;
Rui-Bo LI
;
Zhi-Gang WU
;
Min CHEN
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Cloning, Molecular;
DNA-Binding Proteins;
chemistry;
genetics;
isolation & purification;
metabolism;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
chemistry;
genetics;
metabolism;
Molecular Weight;
Plant Proteins;
chemistry;
genetics;
isolation & purification;
metabolism;
Recombinant Proteins;
chemistry;
genetics;
isolation & purification;
metabolism;
Salvia miltiorrhiza;
genetics
- From:
China Journal of Chinese Materia Medica
2014;39(7):1214-1219
- CountryChina
- Language:Chinese
-
Abstract:
WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.
