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.
Highlights
Staphylococcus aureus is an invasive pathogen that causes various human illnesses, ranging from skin infections and food poisoning to toxic shock syndrome and endocarditis [1]
The secretion of virulence toxins is controlled by the accessory gene regulator quorum sensing system that is composed of an autoinducing peptide (AIP) and a two-component signal transduction system (TCST) [2,3]
To confirm that the reported expression levels and detergent selections are scalable for downstream structure-function applications, the fusion protein AgrC-green fluorescent protein (GFP) was purified by immobilized metal ion affinity chromatography (IMAC) using the best detergent identified at the detergent screening stage (Figure 3A,B)
Summary
Staphylococcus aureus is an invasive pathogen that causes various human illnesses, ranging from skin infections and food poisoning to toxic shock syndrome and endocarditis [1]. The TCST system in S. aureus is comprised of the membrane-bound histidine kinase (HK) sensor AgrC, which responds to the AIP signaling molecule, and the cytoplasmic response regulator (RR) AgrA, which gives rise to a cellular response [8]. The mechanism in S. aureus for sensing and responding to AIP and the specific sites of AgrC oligomer formation remain largely unknown. To resolve these issues at the molecular level and gain insight into the structure of the AgrC, it is important to express and purify a sufficient amount of functional full-length AgrC. We describe, for the first time, a GFP fluorescence-based approach to assess the expression, purification, and detergent solubility of full-length AgrC from the agr-I allele. This study provides a basis for designing rational strategies to improve the yields of membrane protein preparations, and these findings provide the first biochemical data on AgrC
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