丹参转录因子SmWRKY1蛋白的表达和纯化条件优化研究(5)
LIU Yu-zhong， SHEN Ye， RONG Qi-xian， WU Wen-yan， LI Rui-bo，WU Zhi-gang， CHEN Min*
(National Resource Center for Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing 100700， China)
[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·L-1 IPTG at 20 ℃ with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g·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.
[Key words] Salvia miltiorrhiza； SmWRKY1； optimizing expression； inclusion body protein； western blotting
doi：10.4268/cjcmm20140713, http://www.100md.com(刘玉忠 申业 荣齐仙 吴文燕 李瑞博 吴志刚 陈敏)
(National Resource Center for Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing 100700， China)
[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·L-1 IPTG at 20 ℃ with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g·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.
[Key words] Salvia miltiorrhiza； SmWRKY1； optimizing expression； inclusion body protein； western blotting
doi：10.4268/cjcmm20140713, http://www.100md.com(刘玉忠 申业 荣齐仙 吴文燕 李瑞博 吴志刚 陈敏)