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