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.
This is an open access article distributed under the
Creative Commons Attribution License which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
Export to BibTeX
MDPI and ACS Style
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.