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(9): 18470-87, 2013 Sep 06.
Article
em En
| MEDLINE
| ID: mdl-24018890
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Quinases
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Staphylococcus aureus
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Proteínas de Bactérias
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Proteínas de Fluorescência Verde
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Percepção de Quorum
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article