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Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System
Wang, Lina1; Quan, Chunshan2,3; Liu, Baoquan2,3; Wang, Jianfeng2,3; Xiong, Wen2,3; Zhao, Pengchao1; Fan, Shengdi2,3
Source PublicationPLOS ONE
2013-11-26
DOI10.1371/journal.pone.0080400
Volume8Issue:11
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology
WOS SubjectMultidisciplinary Sciences
WOS Research AreaScience & Technology - Other Topics
WOS KeywordSIZE-EXCLUSION CHROMATOGRAPHY ; ESCHERICHIA-COLI ; 2-DIMENSIONAL CRYSTALLIZATION ; PROTEIN RECONSTITUTION ; AUTOINDUCING PEPTIDES ; RECEPTOR ; SENSOR ; DETERGENTS ; LIPOSOMES ; VIRULENCE
AbstractThe integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrC(TM5-6C) and AgrC(TM5-6C)-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrC(TM5-6C) and AgrC(TM5-6C)-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N, N-dimethyldode-cylamine Noxide during complete liposome solubilization, which resulted in a yield of 85 +/- 5%. The COOH-terminus of the protein AgrC(TM5-6C) was almost exclusively oriented towards the inside of the vesicles. AgrC(TM5-6C) in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrC(TM5-6C) in detergent micelles. The reconstitution of AgrC(TM5-6C) or AgrC(TM5-6C)-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.
Language英语
WOS IDWOS:000327546400027
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Cited Times:4[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/137718
Collection中国科学院大连化学物理研究所
Affiliation1.Chinese Acad Sci, Dalian Inst Chem Phys, Dalian, Peoples R China
2.Dalian Nationalities Univ, Dept Life Sci, Dalian, Peoples R China
3.State Ethn Affairs Commiss, Minist Educ, Dalian, Peoples R China
Recommended Citation
GB/T 7714
Wang, Lina,Quan, Chunshan,Liu, Baoquan,et al. Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System[J]. PLOS ONE,2013,8(11).
APA Wang, Lina.,Quan, Chunshan.,Liu, Baoquan.,Wang, Jianfeng.,Xiong, Wen.,...&Fan, Shengdi.(2013).Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System.PLOS ONE,8(11).
MLA Wang, Lina,et al."Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System".PLOS ONE 8.11(2013).
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