A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors 英文参考文献.docVIP

A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors 英文参考文献.doc

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A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors 英文参考文献

ARobustandRapidMethodofProducingSoluble, Stable,andFunctionalG-ProteinCoupledReceptors KarolinaCorin1,PhilippBaaske2,DeepaliB.Ravel1,JunyaoSong1,EmilyBrown1,XiaoqiangWang1,3, SandraGeissler2,ChristophJ.Wienken4,MoranJerabek-Willemsen2,StefanDuhr2,DieterBraun4, ShuguangZhang1* 1CenterforBiomedicalEngineering,MassachusettsInstituteofTechnology,Cambridge,Massachusetts,UnitedStatesofAmerica,2NanoTemperTechnologiesGmbH, Mu¨nchen, Germany, 3Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, Shandong, People’s Republic of China, 4SystemsBiophysics,FunctionalNanosystems,Ludwig-MaximiliansUniversityMu¨nchen,Mu¨nchen,Germany Abstract Membraneproteins,particularlyG-proteincoupledreceptors(GPCRs),arenotoriouslydifficulttoexpress.Usingcommercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at .90% purity. Secondary structure analysis using circular dichroism indicatedthatthepurifiedreceptorswereproperlyfolded.Microscalethermophoresis,anovellabel-freeandsurface-free detectiontechniquethatusesthermalgradients,showedthatthesereceptorsboundtheirligands.Thesecondarystructure andligand-bindingresultsfromcell-freeproducedproteinswerecomparabletothoseexpressedandpurifiedfromHEK293 cells.Ourstudydemonstratesthatcell-freeproteinproductionusingcommerciallyavailablekitsandoptimaldetergentsisa robusttechnologythatcanbeusedtoproducesufficientGPCRsforbiochemical,structural,andfunctionalanalyses.This robustandsimplemethodmayfurtherstimulateotherstostudythestructureandfunctionofmembraneproteins. Citation:CorinK,BaaskeP,RavelDB,SongJ,BrownE,etal.(2011)ARobustandRapidMethodofProducingSoluble,Stable,andFunctionalG-ProteinCoupled Receptors.PLoSONE6(10):e23036.doi:10.1371/journal.pone.0023036 Editor:JianR.Lu,TheUniversityofManchester,UnitedKingdom ReceivedApril2,2011;AcceptedJuly4,2011;PublishedOctober25,2011 Copyright: ? 2011 Corin e

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