Expression,Purification and Identification of AgrA, a Response Regulator Protein of Two-component Signal Transduction System in <em>Staphylococcus aureus</em>

doi:10.13523/j.cb.20150505

China Biotechnology

2015, Vol. 35

Issue (5): 32-40 DOI: 10.13523/j.cb.20150505

Expression,Purification and Identification of AgrA, a Response Regulator Protein of Two-component Signal Transduction System in Staphylococcus aureus

ZHANG Xu-ning¹, QUAN Chun-shan^2,3, LIAO Ying-ling^2,3, LIU Ke-huan^2,3, XIONG Wen^2,3, FAN Sheng-di^2,3

1. School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China;
2. Key Laboratory of Biotechnology and Resource Utilization, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, Dalian 116600, China;
3. Department of Life Science, Dalian Nationalities University, Dalian 116600, China

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Abstract

As a response regulator in the two-component signal transduction system (TCST) of Staphylococcus aureus, AgrA, which transmits signals to downstream promoter and then regulates genes expression and transcription, plays an important role in the pathogenesis of S. aureus. A expression vector using Restriction-free (RF) cloning was constructed. In order to monitor the protein expression level in real time, a C-terminal green fluorescent protein (GFP) was fused to AgrA to serve as a reporter. Firstly, BL21-(DE3)-PlysS was screened as the best host cell by single-factor experiment. Secondly, combined with the Box-Behnken designed response surface method test, the cultured conditions which include culture time、RPM and IPTG concentration were optimized. The optimum conditions selected are as follows: induction time is 22h, speed is 222r/min, IPTG is 0.5mmol/L. Then, the expressed AgrA were purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) with the yield of AgrA is 5.56mg/L. Finally, the activity of AgrA was assessed by using non-radioactive electrophoretic mobility shift assay (EMSA) based on AgrA protein LytTR region. In short, A strategy through which response regulator protein AgrA could be expressed in E. coli efficiently and solublely. It not only establishes the foundation for the two-component signal transduction system in vitro studies, but also provides a viable reference for other response regulator proteins which need soluble expression, separation and purification.

Key words： Staphylococcus aureus AgrA GFP Response surface method EMSA

Received: 22 January 2015 Published: 25 May 2015

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ZHANG Xu-ning, QUAN Chun-shan, LIAO Ying-ling, LIU Ke-huan, XIONG Wen, FAN Sheng-di. Expression,Purification and Identification of AgrA, a Response Regulator Protein of Two-component Signal Transduction System in Staphylococcus aureus. China Biotechnology, 2015, 35(5): 32-40.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150505 OR https://manu60.magtech.com.cn/biotech/Y2015/V35/I5/32

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