superfoler-GFP解决融合蛋白可溶性问题

EngineeringandcharacterizationofasuperfoldergreenfluorescentproteinJean-DenisPe´delacq1,Ste´phanieCabantous1,TimothyTran2,ThomasCTerwilliger1&GeoffreySWaldo1Existingvariantsofgreenfluorescentprotein(GFP)oftenmisfoldwhenexpressedasfusionswithotherproteins.WehavegeneratedarobustlyfoldedversionofGFP,called‘superfolder’GFP,thatfoldswellevenwhenfusedtopoorlyfoldedpolypeptides.Comparedto‘foldingreporter’GFP,afolding-enhancedGFPcontainingthe‘cycle-3’mutationsandthe‘enhancedGFP’mutationsF64LandS65T,superfolderGFPshowsimprovedtoleranceofcircularpermutation,greaterresistancetochemicaldenaturantsandimprovedfoldingkinetics.ThefluorescenceofEscherichiacolicellsexpressingeachofeighteenproteinsfromPyrobaculumaerophilumasfusionswithsuperfolderGFPwasproportionaltototalproteinexpression.Incontrast,fluorescenceoffoldingreporterGFPfusionproteinswasstronglycorrelatedwiththeproductivefoldingyieldofthepassengerprotein.X-raycrystallographicstructuralanalyseshelpedexplaintheenhancedfoldingofsuperfolderGFPrelativetofoldingreporterGFP.Wild-typegreenfluorescentprotein(GFP)misfoldswhenexpressedinE.coli1.Better-foldedvariants1–3ofGFParewidelyemployedasproteinfusiontags1,4–8,butthefusedproteinscanreducethefold-ingyieldandfluorescenceoftheseGFPs9–13.CircularlypermutedvariantsofGFP,aswellasbiosensorsusingfusedreceptordomainsandengineeredGFPs,tendtoaggregateandmisfoldwhenexpressedinE.coli14–16.AmorerobustlyfoldedversionofGFPwouldbeveryuseful.DespiteconsiderableeffortaimedatimprovingthefoldingofGFPexpressedalone1–3,availableGFPvariantsfoldwellandarebrightlyfluorescentonlywhenexpressedaloneorwhenfusedtowell-foldedproteins9–12.TheresistanceoffoldedGFPtochemicalandthermaldenaturation1makestheselectionofevenmorerobustlyfoldedGFPvariantschallenging.Wereasonedthatevenbetter-foldedversionsofGFPcouldbeidentifiedbyexpressinglibrariesofGFPvariantsasfusionswithanN-terminal,poorlyfolded,‘bait’polypeptidethatinterfereswiththeproductivefoldingofthefusedGFPmoiety9,andthenselectingbrighterclonescorrespondingtoGFPvariantsthatcanstillfold.WeusedthisapproachtogenerateaveryrobustlyfoldedversionofGFP,superfolderGFP.Thefluores-cenceofsuperfolderGFPfusionisunaffectedbyfusionpartnermisfoldingandisdirectlyproportionaltototalexpressionregard-lessofthesolubilityofthefusion,makingsuperfolderGFPfluores-cencearobustreporteroffusionproteinexpression.X-raycrystal-lographicstudiesofbothsuperfolderGFPandfoldingreporterGFPrevealafive-memberedion-pairnetworkinthesuperfolderGFPstructure,mediatedbythemutationS30R.ThishelpsexplainwhythemutationS30RsubstantiallyimprovesthefoldingrobustnessofsuperfolderGFP.RESULTSGeneratingfluorescentproteinswithenhancedfoldingrobustnessStartingwiththefoldingreporterGFP9,awell-foldedGFPvariantbearingthe‘cycle-3’mutations17F99S,M153T,V163A,andthe‘enhancedGFP’mutations18F64LandS65T,wescreenedlibrariesoffoldingreporterGFPvariantsasC-terminalfusionstopoorlyfolded,bullfrogred-cell,H-subunitferritin,aninsolubleproteinwhenexpressedaloneinE.coliat371C9.Coloniesexpressingtheferritin–foldingreporterGFPfusionat371Cshowedveryfaintfluorescence9.AfterfourroundsofDNAshufflingduringwhichwepickedeverbrighterfluorescentclonesexpressedat371C,weobtainedthehighlyfluorescent,ferritin–superfolderGFPfusion.SuperfolderGFPcon-tainsthefoldingreportermutationsandsixnewmutations:S30R,Y39N,N105T,Y145F,I171VandA206V(Fig.1).LiquidculturesofE.coliBL21(DE3)cellsexpressingferritin–superfolderGFPfusionswereB50-foldmorefluorescentthancellsexpressingferritin–foldingreporterGFPat371C(Table1).E.colicellsexpressingsuperfolderGFPalonewereapproximatelytwofoldmorefluorescentthancellsexpressingfoldingreporterGFPalone(see‘controls’inTable2).WealsoexpressedsixvariantsoffoldingreporterGFPat271Cor371C,eachbearingoneofthesingle-pointmutationsderivedfromsuperfolderGFPasC-terminalfusionswithferritin(Table1).EachofthemutationsincreasedtheamountoffoldedfluorescentGFPfusedtopoorlyfoldedferritinrelativetothefoldingreporterGFPfusion(Table1).ThefluorescenceemissionspectraofsuperfolderGFPandfoldingreporterGFPweresuper-imposable(SupplementaryFig.1aonline).Fluorescenceexcitationandultraviolet-visiblespectraoffoldingreporterGFPeachpeakedat490nm,whereasthecorrespondingsuperfolderGFPspectrapeakedReceived20June;accepted25August;publishedonline20December2005;correctedafterprint1September2006;doi:10.1038/nbt11721BioscienceDivision,MS-M888,LosAlamosNationalLaboratory,LosAlamos,NewMexico87545,USA.2CornellUniversity,DepartmentofChemistry&ChemicalBiology,P.O.Box305,Ithaca,NewYork14851-0305,USA.CorrespondenceshouldbeaddressedtoG.S.W.([e-mail]).NATUREBIOTECHNOLOGYVOLUME24NUMBER1JANUARY200679ARTICLES©2006NaturePublishingGroup(SupplementaryFig.1a,bonline).TherelativequantumefficienciesoffoldingreporterGFPandsuperfolderGFPwere0.72and0.65,usingtheexperimentallydeterminedmolarextinctioncoefficients8.24
104M–1cm–1and8.33
104M–1cm–1.Super-folderGFPphotobleachedslightlyfasterthanfoldingreporterGFP,losing20–25%oftheinitialfluorescenceafter40mincontinuousilluminationinaplatereader(SupplementaryFig