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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (11): 52-58    DOI: 10.13523/j.cb.20171108
研究报告     
金黄色葡萄球菌arl双组分信号转导系统受体蛋白ArlSCA的表达、纯化及活性研究
王路路1,2, 权春善2,3, 许永斌2,3, 陈金利2,3, 吴懿2,3, 王冠天2,3, 董悦生1
1. 大连理工大学环境与生命学院 大连 116024;
2. 大连民族大学生物技术与资源利用教育部重点实验室 大连 116600;
3. 大连民族大学生命科学学院 大连 116600
Expression, Purification and Functional Assay of ArlSCA from Staphylococcus aureus
WANG Lu-lu1,2, QUAN Chun-shan2,3, XU Yong-bin2,3, CHEN Jin-li2,3, WU Yi2,3, WANG Guan-tian2,3, DONG Yue-sheng1
1. School Environment & Biological Science & Technology, Dalian University of Technology, Dalian 116024, China;
2. Key Laboratory of Biotechnology and Bioresources Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian 116000, China;
3. College of Life Science, Dalian Minzu University, Dalian 116600, China
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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ArlSCA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-ArlSCAG418A和pProEX-HTa-ArlSCAG420A。ArlSCAG418A和ArlSCAG420A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。
关键词： ArlSCA;  蛋白质纯化;  金黄色葡萄球菌;  激酶活性    
Abstract: 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 ArlS 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 ArlS can reach 15mg 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 ArlS 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-ArlSCAG418A and pProEX-HTa-ArlSCAG420A 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 ArlSCAG418A and ArlSCAG420A did not have the kinase activity, illustrating that the amino acid residues at 418 and 420 are crucial to the autophosphorylation process of ArlS.
Key words: ArlSCA    Kinase activity    Staphylococcus aureus    Purification
收稿日期: 2017-04-19 出版日期: 2017-11-15
ZTFLH:  Q816  
基金资助: 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
通讯作者: 权春善     E-mail: mikyeken@dlnu.edu.cn
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引用本文:

王路路, 权春善, 许永斌, 陈金利, 吴懿, 王冠天, 董悦生. 金黄色葡萄球菌arl双组分信号转导系统受体蛋白ArlSCA的表达、纯化及活性研究[J]. 中国生物工程杂志, 2017, 37(11): 52-58.

WANG Lu-lu, QUAN Chun-shan, XU Yong-bin, CHEN Jin-li, WU Yi, WANG Guan-tian, DONG Yue-sheng. Expression, Purification and Functional Assay of ArlSCA from Staphylococcus aureus. China Biotechnology, 2017, 37(11): 52-58.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171108    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I11/52

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