ApplMicrobiolBiotechnol(2003)60:523 - PDF document

ApplMicrobiolBiotechnol(2003)60:523…533DOI10.1007/s00253-002-1158-6 MINI-REVIEWK.TerpeOverviewoftagproteinfusions:frommolecularandbiochemicalfundamentalstocommercialsystems Received:8July2002/Revised:25September2002/Accepted:27September2002/Publishedonline:7November2002
Springer-Verlag2002 Abstract K.Terpe(TechnicalConsultantoftheIBAGmbH,Proteinexpression/purificationandnucleicacids,37079Gttingen,e-mail:terpe@iba-go.deTel.:+49-551-50672121Fax:+49-551-50672181 lized(Bucheretal.2002).Thecrystalswerevisuallyindistinguishablefromcrystalsofthenativeprotein;however,thecrystalsdiddifferinmosaicityanddiffrac-tion.C-terminalseriesofarginineresiduescanberemovedbycarboxypeptidaseBtreatment.Thisenzy-maticprocesshasbeensuccessfullyusedinseveralinstances,butoftenhasbeenlimitedbypoorcleavageyieldsorbyunwantedcleavageoccurredwithinthedesiredproteinsequence(NagaiandThogerson1987).TheArg-tagcanbeusedtoimmobilizefunctionalproteinsonflatsurfaces;thisisimportantforstudyinginteractionswithligands.GFPwithanArg-tagononeofitsterminicanbereversiblyandspecificallyboundviathissequenceontoamicasurface,whichhasbeenestablishedasastandardsubstrateforelectronandscanningprobemicroscopyapplications(Nocketal.1997).WhiletheArg-tagisnotusedveryoften,incombinationwithasecondtagitcanbeaninterestingtoolforproteinpurification.Polyhistidine-tag(His-tag)Awidelyemployedmethodutilizesimmobilizedmetal-affinitychromatographytopurifyrecombinantproteinscontainingashortaffinity-tagconsistingofpolyhistidineresidues.Immobilizedmetal-affinitychromatography(IMAC;describedbyPorathetal.1975)isbasedontheinteractionbetweenatransitionmetalion(Co,Ni,Zn)immobilizedonamatrixandspecificamino-acidsidechains.Histidineistheaminoacidthatexhibitsthestrongestinteractionwithimmobilizedmetalionmatrices,aselectrondonorgroupsonthehistidineimidazoleringreadilyformcoordinationbondswiththeimmobilizedtransitionmetal.Peptidescontainingse-quencesofconsecutivehistidineresiduesareefficientlyretainedonIMAC.Followingwashingofthematrixmaterial,peptidescontainingpolyhistidinesequencescanbeeasilyelutedbyeitheradjustingthepHofthecolumnbufferorbyaddingfreeimidazole(Table1).ThemethodtopurifyproteinswithhistidineresidueswasfirstTable1Matricesandelutionconditionsofaffinitytags AffinitytagMatrixElutionconditionPoly-ArgCation-exchangeresinNaCllineargradientfrom0to400mMatalkaline�pH8.0Poly-HisNi-NTA,Co-CMA(Talon)Imidazole20…250mMorlowpHFLAGAnti-FLAGmonoclonalantibodypH3.0or2…5mMEDTAStrep-tagIIStrep-Tactin(modifiedstreptavidin)2.5mMdesthiobiotinc-mycMonoclonalantibodyLowpHSS-fragmentofRNaseA3Mguanidinethiocyanate,0.2McitratepH2,3MmagnesiumchlorideHAT(naturalhistidineaffinitytag)-CMA(Talon)150mMimidazoleorlowpHCalmodulin-bindingpeptideCalmodulinEGTAorEGTAwith1MNaClCellulose-bindingdomainCelluloseFamilyI:guanidineHClor�urea4MFamilyII/III:ethyleneglycolSBPStreptavidin2mMBiotinChitin-bindingdomainChitinFusedwithintein:30…50mMdithiothreitol,-mercaptoethanolorcysteineGlutathioneS-transferaseGlutathione5…10mMreducedglutathioneMaltose-bindingproteinCross-linkedamylose10mMmaltoseTable2Sequenceandsizeofaffinitytags TagResiduesSequenceSizePoly-Arg5…6(usually5)Poly-His2…10(usually6)HHHHHH0.84FLAG8DYKDDDDK1.01Strep-tagII8WSHPQFEK1.06c-myc11EQKLISEEDL1.20S-15KETAAAKFERQHMDS1.75HAT-19KDHLIHNVHKEFHAHAHNK2.313xFLAG22DYKDHDGDYKDHDIDYKDDDDK2.73Calmodulin-bindingpeptide26KRRWKKNFIAVSAANRFKKISSSGAL2.96Cellulose-bindingdomains27…189DomainsSBP38MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP4.03Chitin-bindingdomain51TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ5.59GlutathioneS-transferase211ProteinMaltose-bindingprotein396Protein describedin1987(Hochulietal.1987).Hochulihasdevelopedanitrilotriaceticacid(NTA)adsorbentformetal-chelateaffinitychromatography.TheNTAresinformsaquadridentatechelateandisespeciallysuitableformetalionswithcoordinationnumbersofsix,sincetwovalenciesremainforthereversiblebindingofbiopoly-mers.Dihydrofolatereductasewithapoly-His-tagwassuccessfullypurifiedwithNi-NTAmatricesin1988(Hochulietal.1988).Thepurificationefficiencyofthissystemwasdependentonthelengthofthepoly-histidineandthesolventsystem(Table3).WhilethesystemworkedefficientlywithHis-taggedproteinsunderdena-turingconditions,His-taggedproteinswereefficientlypurifiedunderphysiologicalconditions.However,HistaggedproteinscanbeboundtoNi-NTAmatricesundernativeconditionsinlow-orhigh-saltbuffers.Afterbinding,thetargetproteincanbeelutedbyanimidazolegradientfrom0.8to250mM.Washingwithalowconcentrationofimidazole(e.g.0.8mM)reducesnon-specificbindingofhostproteinswithhistidines.ElutionofHis-taggedproteinsiseffectivewithinarangeof20…250mMimidazole(Heftietal.2001;Janknechtetal.1991).AdisadvantageofusingimidazoleisthatitcaninfluenceNMRexperiments,competitionstudies,andcrystallographictrials,andthepresenceofimidazoleoftenresultsinproteinaggregates(Heftietal.2001).AnothermaterialthathasbeendevelopedtopurifyHis-taggedproteinsisTALON.ItconsistsofaCocarboxylmethylaspartate(Co-CMA),whichiscoupledtoasolid-supportresin.TALONallowstheelutionoftaggedproteinsundermildconditions,andithasbeenreportedtoexhibitlessnon-specificproteinbindingthantheNi-NTAresin,resultinginhigherelutionproductpurity(Chagaetal.1999a,b).Afinalpreparationofenzymesexhibitedapurityhigherthan95%asascer-tainedbySDS-PAGE.PurificationwithCoallowedthedevelopmentofanatural19-amino-acidpoly-histidineaffinitytag(HAT-tag;forthesequence,seeTable2).Chloramphenicolacetyltransferase,dihydrofo-latereductase,andgreenfluorescentproteinwithN-terminalHAT-tagswerepurifiedundermildconditionsinonestepwithapurityover95%.Adsorptionofweaklyboundunspecificproteinswaseliminatedbyusing5mMimidazoleintheequilibrationandloadingbuffer,and150mMimidazolewasusedtoelutetheHAT-taggedproteins.ElutionoftaggedproteinswasalsopossiblebydecreasingthepHto5.0.UreaturnedouttohaveamuchstrongernegativeeffectonthebindingofHAT-taggedproteinsthanguanidiniumHCl.However,over-expres-sionwithHAT-taghasonlybeentestedinbacteria.Poly-histidineaffinitytagsarecommonlyplacedoneithertheN-ortheC-terminusofrecombinantproteins.Optimalplacementofthetagisprotein-specific.Purifi-cationusingpoly-histidinetagshasbeencarriedoutsuccessfullyusinganumberofexpressionsystemsincludingbacteria(ChenandHai1994;Ranketal.2001),yeast(Borsingetal.1997;KaslowandShiloach1994),mammaliancells(Janknechtetal.1991;JanknechtandNordheim1992),andbaculovirus-infectedinsectcells(Kuusinenetal.1995;Schmidtetal.1998).Morethan100structuresofHis-taggedproteinshavebeendepositedintheProteinDataBank.ProteinswithaHis-tagmayvaryslightlyasfarastheirmosaicityanddiffractioncomparedtothenativeprotein(Hakanssonetal.2000).Inprinciple,itcannotbeexcludedthattheaffinitytagmayinterferewithproteinactivity(WuandFilutowicz1999),althoughtherelativelysmallsizeandchargeofthepolyhistidineaffinitytagensurethatproteinactivityisrarelyaffected.Movingtheaffinitytagtotheoppositeterminus(Halliwelletal.2001)orcarryingoutthepurificationunderdenaturingconditionsoftensolvesthisproblem.PurificationofproteinwithametalcenterisnotrecommendedbecausethemetalcanbeabsorbedbytheNTA.PurificationunderanaerobicconditionsisalsonotrecommendedbecauseNi-NTAisreduced.Never-theless,purificationofproteinswithHis-tagisthemostcommonlyusedmethod.TheFLAG-tagsystemutilizesashort,hydrophilic8-amino-acidpeptide(Table1)thatisfusedtotheproteinofinterest(Hoppetal.1988).TheFLAGpeptidebindstotheantibodyM1.Whetherbindingiscalcium-dependentmanner(Hopeetal.1996)or-independent(EinhauerandJungbauer2000)remainscontroversial.KineticstudiesforbindingofFLAG-GFP,evaluatedbyBIACORETable3Affinityofpolyhisti-dinedihydrofolatereductase(DHFR)fortheNi-NTAad-sorbentin6Mguanidinehy-drochloride(GuHCl)and0.05Mphosphatebuffer(Hochulietal. PhosphateGuHClRetained(%)Eluted(%)Retained(%)Eluted(%)Polyhistidinedihydrofolatereductase-DHFR3010…-DHFR9075…-DHFR-5;܉.;　90301010-DHFR-5;܉.;　90205050-DHFR-5;܉.;　9010-5;܉.;　9090-5;܉.;　9090…-57; .8;9080…-5;܉.;　90501010-5;܉.;　90405050-5;܉.;　9030-5;܉.;　9090 analysis,wereidenticalinthepresenceandabsenceofions.Additionaltargetsarethemonoclonalantibod-iesM2andM5,eachwithdifferentrecognitionandbindingcharacteristics.TheFLAG-tagcanbelocatedattheC-orN-terminusoftheprotein.Thesystemhasbeenusedinavarietyofcelltypes,includingexamplesfrombacterial(BlanarandRutter1992;Suetal.1992),yeast(Einhaueretal.2002;Schusteretal.2000),andmammaliancells(Kunzetal.1992;Zhangetal.1991).Thepurificationconditionofthesystemisnon-denaturingandthusallowsactivefusionproteinstobepurified.ThecomplexcanbedissociatedbychelatingagentssuchasEDTAorbytransientlyreducingthepH(Table1).Adisadvantageofthesystemisthatthemonoclonal-antibodypurificationmatrixisnotasstableasothers,e.g.Ni-NTAorStrep-Tactin.Thepurityofisolatedproteinsisintherangeof90%(Schusteretal.2000).Ingeneral,smalltagscanbedetectedwithspecificmono-clonalantibodies.ToimprovethedetectionoftheFLAG-tagthe3xFLAGsystemhasbeendeveloped.Thisthree-tandemFLAGepitopeishydrophilic,22-amino-acidslong(Table2)andcandetectupto10fmolofexpressedfusionprotein.TheFLAG-taggedmaltodextrin-bindingproteinofPyrococcusfuriosushasbeencrystallized(Bucheretal.2002)andthequalityofthecrystalswasverysimilartothatofcrystalsofuntaggedprotein.Finally,theFLAG-tagcanberemovedbytreatmentwithenterokinase,whichisspecificforthefiveC-terminalaminoacidsofthepeptidesequence(Marouxetal.1971).TheStrep-tagisanaminoacidpeptidethatwasdevelopedasanaffinitytoolforthepurificationofcorrespondingfusionproteinsonstreptavidincolumns(SchmidtandSkerra1993).Streptavidinmutantswithaspecificmutationatposition44,45,and47haveahigheraffinityfortheoctapeptideStrep-tagIIthanforthenativeform(forthesequence,seeTable2;Schmidtetal.1996;VossandSkerra1997;KorndrferandSkerra2001).ThisstreptavidinvariantiscalledStrep-Tactin.Strep-taggedproteinsareboundunderphysiologicalbufferconditionsinthebiotinbindingpocket,andcanbeelutedgentlywithbiotinderivatives.Elutionwith2.5mMdesthiobiotinisrecommended.Thematrixcanberegeneratedwith4-hydroxyazobenzene-2-carboxylicacid,whichisyellowinsolutionandredwhenboundonthematrix.Thebindingconditionsareveryspecific.BiotinylatedproteinssuchasthecarboxylcarrierproteinofEscherichiacoliarealsoboundonStrep-Tactin,butbiotinorbiotinylatedproteinscanbeblockedwithavidin.Thepurificationconditionsarehighlyvariable.Chelatingagents,milddetergents,reductiondetergents,andsaltupto1Mcanbeaddedtothebuffer.Denaturingpurificationconditions,such6Murea,destroytheStrep-tag/Strep-Tactininter-actionbutnotStrep-Tactin.TheinteractionbetweenthetagandStrep-Tactinisclosetotherangeof1M(VossandSkerra1997).FusionproteinscanbespecificallydetectedbyStrep-Tactinconjugatesorbyantibodies.ThetagcanbeengineeredtoeithertheC-orN-terminusofaprotein.RecombinantStrep-tag-hybridsareproducedinbacteria(Fontaineetal.2002),yeast(MurphyandLagarias1997),mammaliansystems(Srdyetal.2002;Smythetal.2000),plants(Druckeretal.2002)andbaculovirus-infectedinsectcells.Thismethodisrecom-mendedforpurifyingactivefusionproteinswithasmalltagunderanaerobicconditions(HansandBuckel2000;Judaetal.2001),andformetal-containingenzymes.Integrationoftaggedproteinsintothemembraneisalsopossible(Großetal.2002).Membraneproteinsubunitswithnotagcouldbeco-purified.Aspecialapplicationofthetagisthatitcanbeusedforeukaryoticsurfacedisplay(Ernstetal.2000).ThecompatibilityofStrep-TactinbindingbiotinandStrep-tagwasusedtoobservetherotatingc-subunitoligomerofEF-F-ATPase(Pnkeetal.2000).TheuseofStrep-taghaswidelyincreasedduringthelastyears.RecombinantproteinswiththetagcanbeusedforNMRandcrystallization(Ostermeieretal.1997).TheStrep-tagsystemisofrelevanceforstudiesonprotein-proteininteractionandspecialapplicationsinwhichlargeorchargedtagsarenotfunctional.Themurinanti-c-mycantibody9E10wasdevelopedin1985(Evanetal.1985)andisusedasanimmunochem-icalreagentincellbiologyandinproteinengineering.Theantibodyepitopeofelevenaminoacids(Table2)canbeexpressedinadifferentproteincontextandstillconfersrecognitionbythe9E10immunoglobulin(MunroandPelham1986).Thec-myc-taghasbeensuccessfullyusedinWestern-blottechnology,immunoprecipitation,andflowcytometry(Kipriyanov1996).Itisthereforeusefulformonitoringexpressionofrecombinantproteinsinbacteria(Dreheretal.1991;Vaughanetal.1996),yeast(Sequi-Realetal.1995;Weissetal.1998),insectcells(Schiothetal.1996),andmammaliancells(McKern1997;MoorbyandGherardi1999).Thesuccessfulco-immunopurificationofinteractingproteinsexpressedincellswasalsoreported(Ferrandoetal.2001).c-myc-taggedproteinscanbeaffinity-purifiedbycouplingMab9E10todivinylsulphone-activatedagarose.ThewashingconditionsarephysiologicalfollowedbyelutionatlowpH,whichcouldexertanegativeeffectonproteinactivity.Purifiedc-myc-taggedproteinshavebeencrystallized(McKernetal.1997).Thec-myc-tagcanbeplacedattheN-orC-terminus(Mansteinetal.1995).Itisawidelyuseddetectionsystembutisrarelyappliedforpurifications.TheS-tagsequenceisafusion-peptidetagthatallowsdetectionbyarapid,sensitivehomogeneousassayorbycolorimetricdetectioninWesternblots.Thesystemis basedonthestronginteractionbetweenthe15-amino-acidS-tag(Table2)andthe103-amino-acidS-protein,bothofwhicharederivedfromRNaseA(Karpeiskyetal.1994;KimandRaines1994).TheS-protein/S-tagcom-plexhasaof~0.1MwhichdependsonpH,temperature,andionicstrength(Connellyetal.1990).Thetagiscomposedoffourcationic,threeanionic,threeunchargedpolar,andfivenon-polarresidues.ThiscompositionmakestheS-tagsoluble.TheS-tagrapidassayisbasedonthereconstitutionofribonucleolyticactivity.TaggedproteinscanbeboundonS-proteinmatrices.Theelutionconditionsareveryharsh,e.g.bufferwithpH2(Table1);however,itisrecommendedtocleavethetagwithproteasetogetfunctionalproteins.Thesystemisfunctionaltopurifyrecombinantproteinsfrombacteria(LellouchandGeremia1999),mammaliancells,andbaculovirus-infectedinsectcellextracts.Thesystemisoftenusedtogetherwithasecondtag.ThediscoveryofahypersensitivefluorogenicsubstrateforRNaseAmakesthesysteminterestingfordetectionincombinationwithhigh-throughputscreening(Kelemenetal.1999).Calmodulin-bindingpeptidePurificationoffusionproteinscontainingcalmodulin-bindingpeptidewasfirstdescribedin1992(Stofko-Hahnetal.1992).Thepeptidehas26residues(forthesequence,seeTable2)derivedfromtheC-terminusofskeletal-musclemyosinlight-chainkinase,whichbindscalmodulinwithnanomolaraffinityinthepresenceof0.2mMCaCl(Blumenthaletal.1985)Thetightbindingallowsmorestringentwashingconditions,ensuringthatfewcontaminatingproteinswillbeco-purifiedwiththefusionprotein.AsecondelutionstepwithEGTAand1MNaClisusefuliftheproteindoesnotelutecompletelyatthefirststep.ThesystemhasahighspecificitytopurifyrecombinantproteinsinE.colibecausetherearenoendogenousproteinsthatinteractwithcalmodulin.Re-coveryoffusionproteinsis80…90%.Reducingagentsanddetergentsinamountsupto0.1%arecompatiblewiththesystem(Vaillancourtetal.2000).Purificationineukary-oticcellsisnotrecommendedbecausemanyendogenousproteinsinteractwithcalmodulininacalcium-dependentmanner(Head1992).Acalmodulin-bindingpeptidethrombinfusiontagisanexcellenttargetforisotopiclabelingwithwith32]ATPusingproteinkinaseA(Vailan-courtetal.2000).His-taggedproteinkinasecanberemovedbyNi-NTAchromatography.Thisallowsstudiesofproteininteractionorscreeningofbacterio-phageexpressionlibraries.Thecalmodulin-bindingpep-tidecanbeplacedattheN-orC-terminus.TheN-terminallocationmayreducetheefficiencyoftranslation,whilecalmodulin-bindingpeptideattheC-terminuscanresultinhighexpressionlevels(Zhengetal.1997).Cellulose-bindingdomainMorethan13differentfamiliesofproteinswithcellulose-bindingdomains(CBDs)havebeenclassified.CBDscanvaryinsizefrom4to20kDa;theyoccuratdifferentpositionswithinpolypeptides:N-terminal,C-terminalandinternal.SomeCBDsbindirreversiblytocelluloseandcanbeusedforimmobilizationofactiveenzymes(Xuetal.2002);othersbindreversiblyandaremoreusefulforseparationandpurification.CBDsoffamilyIbindreversiblytocrystallinecelluloseandareausefultagforaffinitychromatography.HydrogenbondformationandvanderWaalsinteractionarethemaindrivingforcesforbinding(Tommeetal.1998).Theadvantageofcelluloseisthatitisinert,haslownon-specificaffinity,isavailableinmanydifferentforms,andhasbeenapprovedformanypharmaceuticalandhumanuses.CBDsbindtocelluloseatamoderatelywidepHrange,from3.5to9.5.ThetagcanbeplacedattheN-orC-terminusofthetargetprotein.Theaffinityofthetagissostrongthatanimmobilizedfusionproteincanonlybereleasedwithbufferscontainingureaorguanidinehydrochloride.Thisdenaturatingelutionconditionsmakerefoldingofthefusedtargetproteinnecessary.FusedproteinswithCBDsoffamiliesIIandIIIcanbeelutedgentlyfromcellulosewithethyleneglycol(McCormickandBerg1997).Thislow-polaritysolventpresumablydisruptsthehydrophobicinteractionatthebindingsite.Ethyleneglycolcanberemovedeasilybydialysis.RecombinantCBD-hybridshavebeenproducedinbacteria,yeast,mammaliancells,andbaculovirus-infectedinsectcells(Tommeetal.1998).TheSBP-tagisanewstreptavidin-bindingpeptideandhasalengthof38aminoacids(forthesequence,seeTable2;Wilsonetal.2001).Thedissociationconstantofthetagtostreptavidinis2.5nM.SBP-taggedproteinscanbepurifiedwithimmobilizedstreptavidin.Theelutionconditionsareverymild,using2mMbiotin.ProteinswithC-terminalSBP-taggedproteinswereexpressedinbacteriaandsuccessfullypurified(Keefeetal.2001).Littleisknownregardingfurtherapplications,butthetagseemstobeaninterestingtooltoimmobilizeproteinsonstreptavidin-coatedchips.Chitin-bindingdomainThechitin-bindingdomainfromBacilluscirculanssistsof51aminoacids(Watanabeetal.1994).Theaffinitytagiscommonlyavailableincombinationwithself-splicinginteins.TheinteinfromtheVMA1gene,whichconsistsof454aminoacids,isoftenused(Chongetal.1996,1997).Other,shorterinteinshavealsobeenemployed(Xuetal.2000).Self-cleavageofthethioesterbondcanbeinducedbythiolreagents,suchas1,4-dithiothreitolor ethanol(Table2).TheC-orN-terminalaminoacidresidueofthetargetproteinhasaneffectoninvivoandinvitrocleavage(Xuetal.2000).Ahighsaltconcentrationortheuseofnon-ionicdetergentscanbeemployedtoreducenon-specificbinding,thusincreasingpurity.Theuncleavedfusionprecursorandtheinteintagremainboundtothechitinresinduringtargetproteinelutionandcanbestrippedfromtheresinby1%SDSor6MguanidineHCl.ProteinswithC-orN-terminalchitin-bindingdomainsfusedwithinteinshavebeenexpressedinbacterialsystems(CantorandChong2001;Swedaetal.2001;Wieseetal.2001).GlutathioneS-transferase-tagSingle-steppurificationofpolypetidesasfusionswithglutathioneS-transferase(GST)wasfirstdescribedin1988(SmithandJohnson1988).A26-kDaGSTofSchistosomajaponicum(Tayloretal.1994)wasclonedinE.coliexpressionvector.Fusionproteinscouldbepurifiedfromcrudelysatebyaffinitychromatographyonimmobilizedglutathione.Boundfusionproteinscanbeelutedwith10mMreducedglutathioneundernon-denaturingconditions.Inthemajorityofcases,fusionproteinsaresolubleinaqueoussolutionsandformdimers.TheGST-tagcanbeeasilydetectedusinganenzymeassayoranimmunoassay.Thetagcanhelptoprotectagainstintracellularproteasecleavageandstabilizetherecombinantprotein.InsomecasesGSTfusionproteinsaretotallyorpartlysoluble.Itremainsunclearwhichfactorsareresponsibleforinsolubility,butinseveralinstancesinsolubilityofGSTfusionproteinswasasso-ciatedwiththepresenceofhydrophobicregions.Otherinsolublefusionproteinseithercontainmanychargedresiduesorarelargerthan100kDa.Insomecasesinsolublefusionproteinscanbepurifiedbyaffinitychromatographyiftheyaresolubilizedin1%TritonX-100,1%Tween,10mMdithiothreitol,0.03%SDSor1.5%sarcosylbuffer(FrangioniandNeel1993).Sarcosylinhibitsco-aggregationofproteinswithbacterialoutermembranecomponents.Purificationofotherinsolubleproteinsmustbedonebyconventionalmethods.ItisrecommendedtocleavetheGST-tagfromfusionproteinsbyasite-specificproteasesuchasthrombinorfactorXThePreScissionproteasecontainsthehumanrhinovirus3CproteaseincludingtheGST-tag;theGSTcarrierandtheproteasecanberemovedafterproteolysisbyaffinitychromatographyongluthatione-agarose.TheGST-tagcanbeplacedattheN-orC-terminusandcanbeusedinbacteria(SmithandJohnson1988),yeast(Luetal.1997),mammaliancells(Rudertetal.1996),andbaculovirus-infectedinsectcells(Beekmanetal.1994).GSTfusionproteinshavebecomeabasictoolforthemolecularbiologist.Theyarealsocommonlyusedinstudiesonprotein-DNAinteractions(Beekmanetal.1994;Lassaretal.1989),protein-proteininteractions(Mayeretal.1991;RonandDressler1992)andasantigensforimmunologyorvaccinationstudies(McTigueetal.1995).Maltose-bindingproteinThe40-kDamaltose-bindingprotein(MBP)isencodedbytheEgeneofE.coliK12(Duplayetal.1988).VectorsthatfacilitatetheexpressionandpurificationofforeignpeptidesinE.colibyfusiontoMPBwerefirstdescribedin1988(DiGuanetal.1988).Fusedproteinscanbepurifiedbyone-stepaffinitychromatographyoncross-linkedamylose.Boundfusionproteinscanbeelutedwith10mMmaltoseinphysiologicalbuffer.Bindingaffinityisinthemicro-molarrange.Somefusionproteinsdonotbindefficientlyinthepresenceof0.2%TritonX-100or0.25%Tween20,whileotherfusionsareunaffected.BufferconditionsarecompatiblefrompH7.0…8.5,andupto1Msalt.Denaturingagentscannotbeused.MBPcanincreasethesolubilityofover-expressedfusionproteinsinbacteria,especiallyeukary-oticproteins(SachdevandChirgwin1999).AspacersequencecodingfortenasparagineresiduesbetweentheMBPandtheproteinofinterestincreasesthechancesthataparticularfusionwillbindtightlytotheamyloseresin.TheMBP-tagcanbeeasilydetectedusinganimmuno-assay.Itisnecessarytocleavethetagwithasite-specificprotease.TheMBPcanbefusedattheN-orC-terminusoftheproteiniftheproteinsareexpressedinbacteria(SachdevandChirgwin2000).N-terminallocationcanreducetheefficiencyoftranslation.TheMBPsystemiswidelyusedincombinationwithasmallaffinitytag(Hamiltonetal.2002;PodmoreandReynolds2002).NusA,TrxAandDsbAOnedisadvantagewhenheterologousproteinsarepro-ducedinE.coliisthatproteinsfrequentlyaggregatesasinsolublefoldingintermediates,knownasinclusionbodies.Inordertorecoveranactiveprotein,itmustbesolubilizedwithdenaturingagentssuchas8Mureaor6Mguanidinehydrochloride.OnepossibilitytoavoidinclusionbodiesistouselargeaffinitytagssuchasGSTorMBP.Hydrophilictags,suchastranscriptiontermina-tionanti-terminationfactor(NusA),E.coli(TrxA),orproteindisulfideisomeraseI(DsbA)canincreasesolubility.Adisadvantageis,however,thatproteinswiththesetagscannotbepurifiedwithaspecificaffinitymatrix.Thefusionconstructmustbeusedincombinationwithasmallaffinitytagforpurification.Especially,theNusAproteinincreasesthesolubilityoffusionproteins(Davisetal.1999).Usually,Ecoliproteinpromoteshairpinfoldingandtermination(Gusar-ovandNudler2001).SomeinsolubleproteinsexpressedE.coliremainedsolublewhentaggedN-terminalwithNusA.NusAhasoftenbeenusedincombinationwiththeHis-tag(Harrisson2000).Thioredoxincanbefusedtotheaminoorcarboxylterminusoftheproteinofinterest(Kattietal.1990;LaVallieetal.2000),buttypicallythesequenceisplacedatthe5'end.DsbAincreasesthesolubilityofthetargetproteininthecytoplasmandperiplasmofE.coli.Itisrecommendedtocleavefusion proteinswithNusA,TrxAorDsbAbyasite-specificprotease;thecleavagesitecanbeusedaslinkerpeptide.Othertag-systemsTherearealsoothertagsystemsinuse,whicharenotdescribedindetailinthisreview:StaphylococcalproteinAgenefusionvectorsweredevelopedtopurifyrecombinantproteinsbyIgGaffinitychromatography(Uhlnetal.1983;Nilssonetal.1985).Thisproteiniswell-suitedforaffinitypurificationduetoitsspecificbindingtotheFcpartofimmunoglobulinsofmanyspeciesincludinghuman.AnalogouslytoproteinA,proteinGfromstrainG148canbeusedinthesamemannerbecauseitbindstheFcportionofIgG(Gowardetal.1990).Biotinylationofproteinsusingsmallpeptidetagsarecommonlyusedfordetection,immobilization,andpurification(Cronan1990).Differenttags,suchtheAviTag,PinPointXproteinpurificationsystem,andBio-tag(Schatz1993;TuckerandGrissham-mer1996),havebeendescribed.ThebacteriophageT7andV5epitopesareinterestingtagsforsensitivedetec-tion.Otherepitopetagsfordetectionare:ECS(entero-kinasecleavagesite),HA(hemaglutininA),andGlu-Glu.CleavageofthetagThepresenceofaffinitytagsmayaffectimportantcharacteristicsorfunctionsoftheproteintobestudied.Removalofthetagfromaproteinofinterestcanbeaccomplishedwithasite-specificprotease,andcleavageshouldnotreduceproteinactivity.Removaloftheproteaseaftercleavageiseasierusingarecombinantproteasewithanaffinitytagorusingabiotinylatedprotease.AbiotinylatedproteasecanbedirectlypurifiedduringaffinitychromatographyusingStrep-tag/Strep-Tactinchromatography,orinasecondstepwithstrep-tavidin.Cleavageofthetagwithoutusingaproteaseisalsopossiblebyintroducingaself-splicingintein(Xuetal.2000).Themostcommonlyusedproteasesare:enterokinase,tobaccoetchvirus(TEV),thrombin,andfactorX.Recoveryofthetargetproteindependsonthecleavageefficiency.EnterokinaseisoftentheproteaseofchoiceforN-terminalfusions,sinceitspecificallyrecognizesafive-amino-acidpolypeptide(D-D-D-D-K-X)andcleavesatthecarboxylsiteoflysine.Sporadiccleavageatotherresidueswasobservedtooccuratlowlevels,dependingontheconformationoftheproteinsubstrate(Choietal.2001).Themolecularweightofthelight-chainofenterokinaseis26.3kDa.Oneunitisdefinedastheamountofenterokinasethatwillcleave95%of50gofafusionproteinin8hat23C.BiochemicalanalyseshaveshownthatthecleavageefficiencydependsontheaminoacidresidueXdownstreamoftheDKrecognitionsite(Table4;HosfieldandLu1999).Incontrasttoothertags,theFLAG-tag(DYKDDDK)hasaninternalrecognitionsiteoftheenterokinase.TEVproteaseisasite-specificproteasethathasaseven-amino-acidrecognitionsite.ThesequenceisE-X-X-Y-X-Q-S,andcleavageoccursbetweentheconservedglutamineandserine(Doughertyetal.1989).Xcanbevariousaminoacidresiduesbutnotallaretolerated.TheoptimalsequenceforcleavageisE-N-L-Y-F-Q-S(Car-ringtonandDougherty1988;Dougheryetal.1988).BestresultswillbeobtainedwhentheTEVproteaserecogni-tionsiteisplacedbetweentwodomains.Whencleavageisnotoptimal,insertionofshortlinkersequenceintro-ducingstructuralflexibilitycanimproveefficiency.Thehighspecificity,itsactivityonavarietyofsubstrates,andtheefficientcleavageatlowtemperaturemakesTEVproteaseanidealtoolforremovingtagsfromfusionproteins(Parksetal.1994).TheefficiencyofcleavageisdependentonboththetagandtheproteinfusedtothecarboxylterminusoftheTEVcleavagesite.Thrombinisaproteasewidelyusedtocleavetags.Cleavagecanbecarriedoutattemperaturesbetween20and37Cfor0.3…16h.IncontrasttoenterokinaseandfactorXa,thrombincleavageresultsintheretentionoftwoaminoacidsontheC-terminalsideofthecleavagepointofthetargetprotein.Theoptimalcleavagesitefor-thrombinhasthestructuresofXX1'-X2',whereXandXarehydrophobicaminoacidandX',Xarenon-acidicaminoacids(Chang1985;Changetal.1985;HaunandMoos1992).SomefrequentlyusedrecognitionsitesareL-V-P-R-G-S,L-V-P-R-G-F,andM-Y-P-R-G-N.CleavagebetweenX'ismoreefficientthancleavagebetweenX'.OthershortrecognitionsitesareXX1',whereXorXareglycine.ExamplesareA-R-GandG-K-A,whereTable4Cleavage(%)ofenterokinasethroughdensitometry(HosfieldandLu1999)basedontheaminoacidresidueX.The-ADQLTEEQIA-...ofaGST-cal-modulinfusionproteinwastestedusing5mgproteindigestedwith0.2Uofenterokinasefor16hat37C AminoacidinpositionXCleavageofenterokinase(%)Alanine88Methionine86Lysine85Leucine85Asparagine85Phenylalanine85Isoleucine84Asparticacid84Glutamicacid80Glutamine79Valine79Arginine78Threonine78Tyrosine78Histidine76Serine76Cysteine74Glycine74Tryptophan67Proline61 cleavageoccursafterthesecondresidue.FiveglycineresiduesbetweenthethrombincleavagesiteandtheN-terminaltagenhancethecleavage(GuanandDixon1991).UsingthisglycinekinkerŽ,lessenzymeisnecessarytoeffectcompletedigestion,andinappropriatecleavage,whereitoccurs,maybeavoided.EffectivedigestionwascarriedoutwithpureTrisbuffer,pH8.NaClinthebufferhasaninhibitoryeffect(HaunandMoos1992).Thrombincanberemovedfromthecleavedproductbyaffinitypurificationon-aminoagarose,gelfiltrationwithasuperose-12FPLCcolumn(Yuetal.1995)orbenzamidinesepharose.AfactorXrecognitionsitebetweenthetagandaproteinofinterestcanbeausefultooltocompletelyremoveN-terminalaffinitytags.FactorXcleavesatthecarboxylsideofthefour-amino-acidpeptideI-E[D]-G-R-(NagaiandThogerson1984),whereXcanbeanyaminoacidexceptarginineandproline.Cleavagecanbecarriedoutattemperaturesrangingfrom4to25C.ThepredominantformoffactorXhasamolecularweightofapproximately43kDa,consistingoftwodisulfide-linkedchainsofapproximately27kDaand16kDa.OnSDS-PAGE,thereducedchainshaveapparentmolecularweightsof30kDaand20kDa.Cleavageofthetagbyasite-specificproteasesuchasfactorXhassometimesbeenineffective,andnon-specificdigestionhasbeenreportedusingfactorX(Koetal.1994).Thereasonscanbeinsolubilityoffusionproteinsorthepresenceofdenaturingreagents.Cleavagecanalsobeincreasedbyintroducingapolyglycineregionoffiveaminoacids(RodriguezandCarrasco1995).Dansyl-glu-gly-arg-chloromethylketoneirreversiblyinactivates95%offactorXactivityin1minatroomtemperature.AlthoughfactorXhasbeenlesspopularbecausecleavagerequireslongerincubationtimeandislesseffective,thereareseveralexamplesofitssuccessfuluse(PryorandLeitingAffinitytagsareimportantinproteinpurification.Theycanbehelpfulforstabilizingproteinsorenhancingtheirsolubility.Affinitychromatographyusuallyresultsin90…99%purity.Thechoiceofthepurificationsystemdependsontheproteinitselfandthefurtherapplications.Sometimesthefusedproteincannotbepurifiedbecausethetagisnotsurface-exposed.Usingdenaturingcondi-tionsorplacingthetagattheotherterminuscansolvethisproblem.Inmanycases,asecondaffinitytagisusedtoincreasethepurityafterasecondaffinitychromatographystep(PryorandLeiting1997;Schiothetal.1996);alternatively,onetagcanbeusedforpurificationandtheotherfordetection(Vaughanetal.1996;Luetal.1997).Iftwodifferenttagsareplacedatoppositetermini,full-lengthproductswillbegeneratedaftertwoaffinitychromatographysteps(Ostermeieretal.1995;SunandBudde1995).Multi-taggingisalsopossible,eachtagbeingsuitableforaspecialapplication.Multi-taggingalsoallowsconsecutivepurificationsteps,resultinginhighpurity.Thesehighlypurifiedproteinsallowprotein-proteininteractionstobemeasured.Associatedproteinscanbeidentifiedusingmassspectroscopy(Honeyetal.2001).Aspecialmulti-tagisthetandemaffinitypurifi-cationtag(TAP;Rigautetal.1999;Puigetal.2001).Itconsistsofaproteinofinterest,acalmodulin-bindingpeptide,aTEVproteasecleavagesite,andproteinAforimmobilization.TheTAPtagallowstherapidpurificationofspecificcomplexes.Theapplicationsoftheprocedurearesimilartothoseoftheyeasttwo-hybridscreen(Fromont-Racineetal.1997).TheTap-tagisatoolforproteomeexploration(Gavinetal.2002).Themethodhasbeentestedinyeastbutshouldbeapplicabletoothercellsororganisms.Manytagswithhighaffinitytotheirbindingpartnerarealsousefultoolstoimmobilizepeptidesorproteinsonsurfaces.Immobilizationofbiologicallyactiveproteinsisimportantforresearchandindustry.Furthermore,theimportanceofaffinity-tagtechnologywillincreaseforuseinpeptide/proteinchipdesign,high-throughputpurification,peptide/proteinli-braries,large-scaleproductionsystems,anddrugdeliveryTheauthorthanksProf.A.SteinbcheltosupportthisreviewandProf.F.Mayerforhisadviceduringpreparationofthemanuscript.BeekmanJM,CooneyAJ,EllistonJF,TsaiSY,TsaiMJ(1994)Arapidone-stepmethodtopurifybaculovirus-expressedhumanestrogenreceptortobeusedintheanalysisoftheoxytocinpromoter.Gene146:285…289BlanarMA,RutterWJ(1992)Interactioncloning:identificationofahelix-loop-helixzipperproteinthatinteractswithc-Science256:1014…1018BlumenthalDK,TakioK,EdelmanA,CharbonneauH,TitaniK,WalshKA,KrebsEG(1985)Identificationofthecalmodulin-bindingdomainofskeletalmusclemyosinlightchainkinase.ProcNatlAcadSciUSA82:3187…3191BorsingL,BergerEG,MalissardM(1997)ExpressionandpurificationofHis-taggedbeta-1,4galactosyltransferaseinyeastandinCOScells.BiochemBiophysResCommunBucherMH,EvdokimovAG,WaughDS(2002)Differentialeffectsofshortaffinitytagsonthecrystallizationofmaltodextrin-bindingprotein.BiolCryst58:392…397CantorEJ,ChongS(2001)Intein-mediatedrapidpurificationofCrerecombinase.ProteinExprPurif22:135…140CarringtonJC,DoughertyWG(1988)Aviralcleavagesitecassette:identificationofaminoacidsequencesrequiredfortobaccoetchviruspolyproteinprocessing.ProcNatlAcadSciUSA85:3391…3395ChagaG,HoppJ,NelsonP(1999a)ImmobilizedmetalionaffinitychromatographyonCo-carboxymethylaspartate-agarosesu-perflow,asdemonstratedbyone-steppurificationoflactatedehydrogenasefromchickenbreast.BiotechnolApplBiochemChagaG,BochkariovDE,JokhadzeGG,HoppJ,NelsonP(1999b)Naturalpoly-histidineaffinitytagforpurificati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