Expression, Purification and Functional Assay of ArlS<sub>CA</sub> from <i>Staphylococcus aureus</i>

doi:10.13523/j.cb.20171108

China Biotechnology

2017, Vol. 37

Issue (11): 52-58 DOI: 10.13523/j.cb.20171108

Expression, Purification and Functional Assay of ArlS_CA from Staphylococcus aureus

WANG Lu-lu^1,2, QUAN Chun-shan^2,3, XU Yong-bin^2,3, CHEN Jin-li^2,3, WU Yi^2,3, WANG Guan-tian^2,3, 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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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-ArlS_CAG418A and pProEX-HTa-ArlS_CAG420A were constructed by the method of site-direted mutation, and proteins of ArlS_CAG418A and ArlS_CAG420A were purified using the metal ion affinity chromatography. The results showed that ArlS_CAG418A and ArlS_CAG420A 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： ArlS_CA Kinase activity Staphylococcus aureus Purification

Received: 19 April 2017 Published: 15 November 2017

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	QUAN Chun-shan
	WANG Guan-tian
	CHEN Jin-li
	WANG Lu-lu
	XU Yong-bin
	WU Yi
	DONG Yue-sheng

Cite this article:

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 ArlS_CA from Staphylococcus aureus. China Biotechnology, 2017, 37(11): 52-58.

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


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