Green Fluorescent Protein GFP-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureusReportar como inadecuado




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1

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhong-shan Road, Dalian 116023, China

2

Department of Life Science, Dalian Nationalities University, Economical and Technological Development Zone, Dalian 116600, China

3

The State Ethnic Affairs Commission-Ministry of Education, Economical and Technological Development Zone, Dalian 116600, China



These authors contributed equally to this work.





*

Author to whom correspondence should be addressed.



Abstract Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein GFP fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography IMAC and size exclusion chromatography SEC at yields of ≥10 mg-L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-23-lauryl-ether Brij-35 was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism CD spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses. View Full-Text

Keywords: membrane protein; detergent screening; GFP; IMAC; SEC; CD spectroscopy membrane protein; detergent screening; GFP; IMAC; SEC; CD spectroscopy





Autor: Lina Wang 1,†, Chunshan Quan 2,3,†,* , Baoquan Liu 2,3, Yongbin Xu 2,3, Pengchao Zhao 1, Wen Xiong 2,3 and Shengdi Fan 2,3

Fuente: http://mdpi.com/



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