title: Green fluorescent protein (GFP)-based overexpression screening and characterization of AgrC, a Receptor protein of quorum sensing in Staphylococcus aureus.
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.
authors.affiliation: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhong-shan Road, Dalian 116023, China. mikyeken@dlnu.edu.cn.
authors.forename: Lina
authors.initials: L
authors.lastname: Wang
authors.forename: Chunshan
authors.initials: C
authors.lastname: Quan
authors.forename: Baoquan
authors.initials: B
authors.lastname: Liu
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Xu
authors.forename: Pengchao
authors.initials: P
authors.lastname: Zhao
authors.forename: Wen
authors.initials: W
authors.lastname: Xiong
authors.forename: Shengdi
authors.initials: S
authors.lastname: Fan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D053038:Quorum Sensing / Q000235:genetics / Q000502:physiology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C531166:AgrC protein, Staphylococcus
references.citation: Protein Sci. 2005 Aug;14(8):2011-7
references.pmid: 15987891
references.citation: Protein Expr Purif. 2008 Nov;62(1):1-8
references.pmid: 18692139
references.citation: FEBS Lett. 2001 Oct 26;507(2):220-4
references.pmid: 11684102
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14371-6
references.pmid: 18796603
references.citation: Methods. 2004 Nov;34(3):364-72
references.pmid: 15325654
references.citation: Nat Biotechnol. 1999 Jul;17(7):691-5
references.pmid: 10404163
references.citation: Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5722-7
references.pmid: 18391190
references.citation: Nat Methods. 2006 Apr;3(4):303-13
references.pmid: 16554836
references.citation: Science. 2005 May 27;308(5726):1321-3
references.pmid: 15919996
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):1924-9
references.pmid: 20080618
references.citation: Anal Biochem. 2008 Nov 1;382(1):69-71
references.pmid: 18682245
references.citation: Annu Rev Microbiol. 2001;55:165-99
references.pmid: 11544353
references.citation: Nucleic Acids Res. 2009 Jul;37(Web Server issue):W465-8
references.pmid: 19429891
references.citation: J Biol Chem. 2008 Apr 4;283(14):8930-8
references.pmid: 18222919
references.citation: Microb Ecol. 2006 Feb;51(2):166-76
references.pmid: 16435167
references.citation: Protein Sci. 2002 Dec;11(12):2774-91
references.pmid: 12441377
references.citation: Chem Rev. 2011 Jan 12;111(1):117-51
references.pmid: 21174435
references.citation: Protein Expr Purif. 2011 Jul;78(1):6-13
references.pmid: 21354311
references.citation: Annu Rev Cell Dev Biol. 2005;21:319-46
references.pmid: 16212498
references.citation: J Microbiol. 2009 Oct;47(5):572-81
references.pmid: 19851730
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):1216-21
references.pmid: 19147840
references.citation: Eukaryot Cell. 2008 Dec;7(12):2017-36
references.pmid: 18952900
references.citation: Annu Rev Genet. 2008;42:541-64
references.pmid: 18713030
references.citation: Methods. 2007 Apr;41(4):388-97
references.pmid: 17367711
references.citation: Nat Methods. 2008 Feb;5(2):135-46
references.pmid: 18235434
references.citation: Nat Protoc. 2008;3(5):784-98
references.pmid: 18451787
references.citation: Annu Rev Genet. 2007;41:121-45
references.pmid: 18076326
references.citation: J Mol Biol. 2008 Aug 29;381(2):300-9
references.pmid: 18582472
references.citation: Biochemistry. 2002 Aug 6;41(31):10095-104
references.pmid: 12146974
references.citation: Protein Expr Purif. 2005 May;41(1):207-34
references.pmid: 15915565
references.citation: Protein Expr Purif. 2003 Jan;27(1):109-14
references.pmid: 12509992
references.citation: Protein Sci. 2009 Jan;18(1):121-33
references.pmid: 19177357