摘要
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 a crucial opportunistic pathogen, and the expression of virulence factors in S. aureus is regulated by various of two component signal transduction systems （TCSTs）. ArlRS TCST has a close relation with the growth and division of bacteria, and the signal transduction of ArlRS TCST is implemented by the autophosphorylation of histidine kinase ArlS. The intracellular domain of AdS is regarded as an important functional domain that regulates the expression of virulence factors. The research focus on the kinase activity and function of the intracellular domain of ArlS. The recombinant plasmids pProEX-HTa-arls and pProEX-HTa-arlr were constructed and the target proteins were overexpressed in E. coli BL21. Then different kinds of isolation technologies such as the metal ion affinity chromatography, the ion exchange chromatography and the gel filtration chromatography were used for purification of the recombinant proteins. The production of ArlR can reach 25mg with about 98% purity from one liter culture medium, and the production of AdS can reach 15rag with about 90% purity from one liter culture medium. The circular dichroism detection results showed that the purified ArlR has natural secondary structure. In vitro phosphorylation results showed that AdS exhibited kinase activity and the ability of autophosphorylation. Furthermore, the phosphorylated ArlS can transfer the phosphate groups to the response regulator protein ArlR. Finally, the recombinant plasmids pProEX-HTa-ArlScAc^lSA and pProEX-HTa- ArlScAc,420A were constructed by the method of site-direted mutation, and proteins of ArlSCAG418A and ArlSCAG420a were purified using the metal ion affinity chromatography. The results showed that ArlSCAcC41sA and ArlSCAC420A did not have the kinase activity, illustrating that the amino acid residues at 418 and 420 are crucial to the autophosphorylation process of AdS.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第11期52-58,共7页
China Biotechnology
基金
国家自然科学基金(21272031
21372037)
中央高校基本科研业务费专项资金(DC201502020201)资助项目
