响应面法优化信号分子AI-2合成蛋白LuxS表达条件

Optimization of expression conditions for signal molecule AI-2 synthetic protein LuxS by response surface methodology

群体感应是一种由自诱导物(autoinducers,AIs)介导的、细菌密度依赖的基因表达调控方式,已经被证实参与细菌多种生理功能的调控.信号分子AI-2介导的LuxS/AI-2型群体感应系统广泛存在于革兰氏阳性和阴性细菌,并备受关注.AI-2的合成依赖于S-核糖同型半胱氨酸酶(LuxS)的催化作用,而LuxS蛋白则是由luxS基因经过转录、翻译得到的.为了探索LuxS蛋白在表达菌株Escherichia coli BL21(DE3)中的最佳表达条件,从而获得较高产量且具有一定的生物活性的LuxS蛋白.首先对LuxS蛋白结构进行预测,确定E.coli BL21(DE3)作为表达菌株.其次,在单因素优化的基础上,进一步通过响应面法优化LuxS蛋白的表达条件.在菌液浓度OD 600为0.5、诱导温度为37℃、IPTG浓度为0.5 mmol/L、诱导时间为31 h条件下,获得LuxS蛋白的最高表达量.研究成功优化了LuxS蛋白在E.coli BL21(DE3)中的诱导表达条件,获得了具有生物活性的LuxS蛋白,并在后续的研究中成功合成了具有生物活性的信号分子AI-2,为体外合成AI-2奠定了基础.

Quorum sensing is a density-dependent gene expression regulation method mediated by autoinducers(AIs)in bacteria,which has been proven to participate in the regulation of multiple physiological functions of bacteria.The LuxS/AI-2 type quorum sensing system mediated by signal molecule AI-2 is widely present in Gram positive bacteria and negative bacteria,which has received considerable attentions.The synthesis of AI-2 depends on the catalytic action of S-ribosylhomocysteinase(LuxS),and the LuxS protein is obtained through transcription and translation of the luxS gene.The purpose of this study is to explore the optimal expression conditions of LuxS protein in the expression strain Escherichia coli BL21(DE3),thereby obtaining a high yield of LuxS protein with certain biological activity.This study first predicted the structure of LuxS protein and determined that E.coli BL21(DE3)was used as the expression strain.Secondly,response surface methodology was further used to optimize the expression conditions of LuxS protein based on single factor optimization.The highest expression condition of LuxS protein was obtained under the conditions of bacterial concentration with OD 600 of 0.5,induction temperature of 37℃,IPTG concentration of 0.5 mmol/L,and induction time of 31 h.This study successfully optimized the expression conditions of LuxS protein in E.coli BL21(DE3),and obtained high yield of LuxS protein with biological activity,laying a foundation for the synthesis of AI-2 in vitro.