金黄色葡萄球菌双组分系统反应调节蛋白AgrA的原核表达、纯化及活性鉴定

Expression,Purification and Identification of AgrA,a Response Regulator Protein of Two-component Signal Transduction System in Staphylococcus aureus

AgrA作为金黄色葡萄球菌双组分信号转导系统(two-component signal transduction system,TCST)的反应调节因子,能调控细菌毒力因子的表达,在金黄色葡萄球菌致病过程中起着重要的作用。采用无限制克隆法构建AgrA表达载体,在AgrA蛋白的C端融合绿色荧光蛋白(GFP)标签,通过实时监测GFP的荧光强度来快速检测重组蛋白的表达水平。首先利用单因素实验,筛选出宿主菌株BL21-(DE3)-PlysS;其次,结合Box-Behnken试验设计,筛选出最优蛋白质表达条件:诱导时间为22h、转速为222r/min、诱导剂浓度为0.5mmol/L,AgrA产量达到5.56mg/L。最后,基于AgrA蛋白LytTR区域的非放射性凝胶阻滞实验(non-radioactive electrophoretic mobility shift assay,EMSA)验证了AgrA的生物活性。提出了反应调节蛋白AgrA在大肠杆菌高效可溶性表达的策略,为双组分信号转导系统的体外研究奠定基础,也为其他反应调节蛋白的可溶性表达与分离纯化提供了一个可行借鉴。

As a response regulator in the two-component signal transduction system （TCST） of Staphylococcus aureus, AgrA, which transmits signals to downstream promoter and then regulates genes expression and transcription, plays an important role in the pathogenesis of S. aureus. A expression vector using Restriction- free （RF） cloning was constructed. In order to monitor the protein expression level in real time, a C-terminal green fluorescent protein （GFP） was fused to AgrA to serve as a reporter. Firstly, BI221-（ DE3 ）-PlysS was screened as the best host cell by single-factor experiment. Secondly, combined with the Box-Behnken designed response surface method test, the cultured conditions which include culture time, RPM and IPTG concentration were optimized. The optimum conditions selected are as follows ： induction time is 22h, speed is 222r/min, IPTG is 0. 5mmol/L. Then, the expressed AgrA were purified using immobilized metal affinity chromatography （IMAC） and size exclusion chromatography （SEC） with the yield of AgrA is 5.56mg/L. Finally, the activity of AgrA was assessed by using non-radioactive eleetrophoretic mobility shift assay （EMSA） based on AgrA protein LytTR region. In short, A strategy through which response regulator protein AgrA could be expressed in E. coli efficiently and solublely. It not only establishes the foundation for the two-component signal transduction system in vitro studies, but also provides a viable reference for other response regulator proteins which need soluble expression, separation and purification.