丹参转录因子SmWRKY1蛋白的表达和纯化条件优化研究  

Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli

作　　者：刘玉忠[1] 申业[1] 荣齐仙[1] 吴文燕[1] 李瑞博[1] 吴志刚[1] 陈敏[1] 

机构地区：[1]中国中医科学院中药资源中心,北京100700

出　　处：《中国中药杂志》2014年第7期1214-1219,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81173490;81130070)

摘　　要：WRKY转录因子是一类含有高度保守的WRKY结构域的锌指蛋白，是植物所特有的转录因子家族。该研究将已克隆的 SmWRKY1 cDNA构建到原核表达载体pET28a上，转化大肠杆菌BL21（DE3）进行诱导表达，SDS-PAGE结果显示该蛋白的大小约36 kDa，与预测的蛋白相对分子质量一样，说明该基因在大肠杆菌中成功表达。对影响蛋白表达的4个因素：诱导时间、诱导温度、IPTG浓度及诱导前菌液的浓度进行优化，结果表明在大肠杆菌BL21（DE3）中，当A600达到约1.0~1.5时，加入0.2 mol·L-1的IPTG，在20 ℃诱导培养12 h后SmWRKY1蛋白的表达量较高。原核表达产物SmWRKY1蛋白以包涵体的形式存在，采用尿素提取包涵体蛋白，用Ni2＋亲和色谱法纯化表达蛋白，纯化后蛋白的质量浓度为2.454 g·L-1。经蛋白质印迹检测，His-Tag单克隆抗体可以特异性的识别SmWRKY1蛋白，进一步证实丹参SmWRKY1蛋白在大肠杆菌中成功表达。本研究为进一步开展 SmWRKY1 基因的表达与丹参酮类等化合物的生物合成相关研究奠定了工作基础。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.

关 键 词：丹参 SmWRKY1 优化表达 包涵体蛋白 蛋白质印迹 

分 类 号：S567.53[农业科学—中草药栽培]