MMB 237 G Protein Signaling, Methods and Protocols

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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME237GProteinGProteinSignalingSignalingMethodsandProtocolsMethodsandProtocolsEditedbyEditedbyAlanV.SmrckaAlanV.SmrckaPurificationofGαfromE.coli31PurificationofRecombinantGProteinαSubunitsfromEscherichiacoliWendyK.G

2、reentreeandMaurineE.LinderSummaryThepurificationofrecombinantGproteinasubunitsexpressedinEscheri-chiacoli(E.coli)isaconvenientandinexpensivemethodtoobtainhomoge-neouspreparationsofproteinforbiochemicalandbiophysicalanalyses.Wild-typeandmutantformsofGαareeasilyproducedfo

3、ranalysisoftheirintrinsicbiochemicalproperties,aswellasforreconstitutionwithreceptors,effectors,regulators,andGproteinβγsubunits.MethodsaredescribedfortheexpressionofGiαandGsαproteinsinE.coli.ProtocolsareprovidedforthepurificationofuntaggedGproteinasubunitsusingconventi

4、onalchromatogra-phyandhistidine(His)-taggedsubunitsusingmetalchelatechromatography.ModificationofGαwithmyristatecanberecapitulatedinE.colibyexpress-ingN-myristoyltransferase(NMT)withitsGproteinsubstrate.ProtocolsfortheproductionandpurificationofmyristoylatedGαarepresent

5、ed.KeyWords:Gprotein;αsubunit;signaltransduction;proteinpurifica-tion;affinitychromatography;GTPase;membraneprotein;myristoylation;N-myristoyltransferase.1.IntroductionHeterotrimericGproteinsarelocalizedattheinnerleafletoftheplasmamembranewheretheyconveysignalsfromcell-

6、surfacereceptorstointracellu-lareffectors(1).Gproteinsfunctionasdimersofanαsubunitandatightlyassociatedβγcomplex.Theαsubunitharborstheguaninenucleotide-bindingsite.Intheinactiveguanosinediphosphate(GDP)-boundstate,Gαisassoci-atedwiththeβγcomplex.ExchangeofGDPforguanosin

7、etriphosphate(GTP)onGαresultsinaconformationalchangethatcausesthesubunitstodissoci-ate.Bothα-GTPandβγinteractwithdownstreameffectorsandregulatetheirFrom:MethodsinMolecularBiology,vol.237:GProteinSignaling:MethodsandProtocolsEditedby:A.V.Smrcka©HumanaPressInc.,Totowa,NJ3

8、4GreentreeandLinderactivity.TheintrinsicGTPhydrolaseactivityoftheαsubunitreturnsthepro-teintotheGDP-boundstate