Liu Yu-Zhong, Shen Ye, Rong Qi-Xian, Wu Wen-Yan, Li Rui-Bo, Wu Zhi-Gang, Chen Min
Zhongguo Zhong Yao Za Zhi. 2014 Apr;39(7):1214-9.
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.
WRKY转录因子是锌指蛋白之一，其含有一个高度保守的WRKY结构域，是植物特异性转录因子家族。携带丹参WRKY1（SmWRKY1）基因的质粒pET28a - SmWRKY1在大肠杆菌BL21（DE3）中成功转化并表达。对大肠杆菌中SmWRKY1的蛋白表达条件进行了优化，包括诱导持续时间、温度、IPTG浓度和大肠杆菌浓度。结果表明，当大肠杆菌在20℃下于0.2 mol·L⁻¹ IPTG存在下培养，A600值为1.0 - 1.5时，培养24小时可获得最高的SmWRKY1蛋白表达量。这种重组的组氨酸标签蛋白以包涵体形式表达，表达量为2.454 g·L⁻¹，首先用尿素提取，然后通过Ni²⁺亲和层析纯化，并通过SDS - PAGE进行鉴定。通过蛋白质免疫印迹分析进一步证实了SmWRKY1在大肠杆菌中的表达。
