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.
Keywords: membrane protein; detergent screening; GFP; IMAC; SEC; CD spectroscopy
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Wang, L.; Quan, C.; Liu, B.; Xu, Y.; Zhao, P.; Xiong, W.; Fan, S. Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus. Int. J. Mol. Sci. 2013, 14, 18470-18487.
Wang L, Quan C, Liu B, Xu Y, Zhao P, Xiong W, Fan S. Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus. International Journal of Molecular Sciences. 2013; 14(9):18470-18487.
Wang, Lina; Quan, Chunshan; Liu, Baoquan; Xu, Yongbin; Zhao, Pengchao; Xiong, Wen; Fan, Shengdi. 2013. "Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus." Int. J. Mol. Sci. 14, no. 9: 18470-18487.