Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

	Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus
	Wang, Lina 1; Quan, Chunshan 2,3; Liu, Baoquan 2,3; Xu, Yongbin 2,3; Zhao, Pengchao 1; Xiong, Wen 2,3; Fan, Shengdi 2,3
刊名	international journal of molecular sciences
	2013-09-01
卷号	14 期号:9 页码:18470-18487
关键词	membrane protein detergent screening GFP IMAC SEC CD spectroscopy
英文摘要	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.
WOS标题词	science & technology ; physical sciences
类目[WOS]	chemistry, multidisciplinary
研究领域[WOS]	chemistry
关键词[WOS]	etch virus protease ; membrane-proteins ; escherichia-coli ; autoinducing peptides ; distinct modes ; purification ; crystallization ; identification ; determinants ; optimization
收录类别	SCI
语种	英语
WOS记录号	WOS:000328623900065
公开日期	2015-11-10
内容类型	期刊论文
源URL	[http://159.226.238.44/handle/321008/137947]
专题	大连化学物理研究所_中国科学院大连化学物理研究所
作者单位	1.Chinese Acad Sci, Dalian Inst Chem Phys, Dalian 116023, Peoples R China 2.Dalian Nationalities Univ, Dept Life Sci, Dalian 116600, Peoples R China 3.Minist Educ, State Ethn Affairs Commiss, Dalian 116600, Peoples R China
推荐引用方式 GB/T 7714	Wang, Lina,Quan, Chunshan,Liu, Baoquan,et al. Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus[J]. international journal of molecular sciences,2013,14(9):18470-18487.
APA	Wang, Lina.,Quan, Chunshan.,Liu, Baoquan.,Xu, Yongbin.,Zhao, Pengchao.,...&Fan, Shengdi.(2013).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,14(9),18470-18487.
MLA	Wang, Lina,et al."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 14.9(2013):18470-18487.