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Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus
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
2013-09-01
DOI10.3390/ijms140918470
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
关键词[WOS]ETCH VIRUS PROTEASE ; MEMBRANE-PROTEINS ; ESCHERICHIA-COLI ; AUTOINDUCING PEPTIDES ; DISTINCT MODES ; PURIFICATION ; CRYSTALLIZATION ; IDENTIFICATION ; DETERMINANTS ; OPTIMIZATION
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.
语种英语
WOS记录号WOS:000328623900065
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被引频次:10[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/137947
专题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
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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.
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