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题名: Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus
作者: Wang, Lina1;  Quan, Chunshan2, 3;  Liu, Baoquan2, 3;  Xu, Yongbin2, 3;  Zhao, Pengchao1;  Xiong, Wen2, 3;  Fan, Shengdi2, 3
关键词: membrane protein ;  detergent screening ;  GFP ;  IMAC ;  SEC ;  CD spectroscopy
刊名: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
发表日期: 2013-09-01
DOI: 10.3390/ijms140918470
卷: 14, 期:9, 页:18470-18487
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Physical Sciences
类目[WOS]: Chemistry, Multidisciplinary
研究领域[WOS]: Chemistry
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.
关键词[WOS]: ETCH VIRUS PROTEASE ;  MEMBRANE-PROTEINS ;  ESCHERICHIA-COLI ;  AUTOINDUCING PEPTIDES ;  DISTINCT MODES ;  PURIFICATION ;  CRYSTALLIZATION ;  IDENTIFICATION ;  DETERMINANTS ;  OPTIMIZATION
语种: 英语
WOS记录号: WOS:000328623900065
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/137947
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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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

Recommended Citation:
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.
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