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金黄色葡萄球菌是一类重要的病原菌,其毒力因子的表达及分泌过程由多种双组分信号转导系统(two component signal transduction system,TCSTS)共同调控,其中ArlRS双组分信号转导系统与细菌的生长和分裂密切相关。ArlRS双组份系统的信号传递通过组氨酸激酶ArlS磷酸化实现,ArlS的胞内域被认为是调控毒力因子表达的重要功能域,以ArlS蛋白的胞内域部分即ArlS_(CA)为目标蛋白进行相关的活性研究。首先,构建pProEX-HTa-arls和pProEX-HTa-arlr重组质粒,对目的蛋白进行诱导表达。其次,利用金属离子螯合层析、离子交换层析以及凝胶过滤层析方法对目的蛋白进行分离纯化,纯化后的ArlR蛋白纯度可达98%,产量约为25mg/L;纯化后的ArlS蛋白纯度可达90%,产量约为15mg/L。圆二色谱检测结果显示纯化后的目的蛋白有完整的二级结构,体外磷酸化结果显示,ArlS蛋白具有激酶活性,自磷酸化后可以将磷酸基团转移给反应调控蛋白ArlR。最后,利用定点突变的方法,构建了418位和420位氨基酸残基突变的表达载体pProEX-HTa-ArlS_(CAG418A)和pProEX-HTa-ArlS_(CAG420A)。ArlS_(CAG418A)和ArlS_(CAG420A)蛋白不具有激酶活性,说明418位和420位氨基酸残基在ArlS蛋白的自磷酸过程中起着关键作用。
Staphylococcus aureus is an important pathogen. The expression and secretion of virulence factors are regulated by a variety of two-component signal transduction system (TCSTS), in which ArlRS two-component signal transduction The system is closely related to the growth and division of bacteria. ArlRS two-component signaling system through the histidine kinase ArlS phosphorylation, ArlS intracellular domain is considered to regulate virulence factor expression of important functional domains to ArlS protein intracellular domain part of that ArlS_ (CA) is The target protein is subjected to related activity studies. First, pProEX-HTa-arls and pProEX-HTa-arlr recombinant plasmids were constructed to express the target protein. Secondly, the target protein was isolated and purified by metal ion chelation chromatography, ion exchange chromatography and gel filtration chromatography. The purity of the purified ArlR protein was 98% and the yield was about 25 mg / L. The purified Arls Protein purity up to 90%, the output is about 15mg / L. The results of circular dichroism showed that the purified target protein had complete secondary structure. The in vitro phosphorylation showed that ArlS protein had kinase activity. After autophosphorylation, the phosphate group could be transferred to the reaction regulatory protein ArlR. Finally, site-directed mutagenesis was used to construct pProEX-HTa-ArlS_ (CAG418A) and pProEX-HTa-ArlS_ (CAG420A) mutants with 418 and 420 amino acid residues. The ArlS_ (CAG418A) and ArlS_ (CAG420A) proteins do not have kinase activity, indicating that the 418 and 420 amino acid residues play a key role in the autophosphorylation of ArlS protein.