| ABSTRACT |
| Glutathione-S-Transferase(GST)fusionproteinshaveawiderangeofapplications.ThisprotocolisdesignedforIPTG-inducIBLebacterialexpressionvectors. |
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| MATERIALS |
| Buffers,Solutions,andReagents |
- Reducedglutathione,20mMpreparedin50mMTris-HCl(pH8.0)
- Isopropyl-β-D-thio-galactoside(IPTG)
- LBbroth,containingappropriateantibioticselection
- Lysisbuffer:PBS+1%TritonX-100,supplementedjustbeforeusewithproteaseinhibitors(e.g,finalconcentrationsof2µg/mlaprotinin,1µg/mlleupeptin,and25µg/mlphenylmethylsulfonylfluoride[PMSF])
- Phosphate-bufferedsaline(PBS),icecold
- 150mMNaCl
- 20mMsodiumphosphate(pH7.4)
- PBSsupplementedjustpriortousewithproteaseinhibitors(e.g.,finalconcentrationsof2µg/mlaprotinin,1µg/mlleupeptin,and25µg/mlPMSF)
- Appropriateantibioticsforplasmidselection(seestep1)
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| BacterialStrains |
- BacterialstraintransformedwithGSTandGSTfusionexpressionplasmids(seenotetostep1)
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| SpecialEquipment |
- Centrifuge,precooledto4°C
- Shakingincubator,presetto37°C
- Rotatorforend-over-endmixing
- Disposablechromatographycolumn
- Glutathione-Sepharosebeads
- Sonicator
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| AdditionalReagents |
- ThisprotocolalsorequiresequipmentandreagentsforSDS-PAGE,includingCoomassieblue(seeSambrookandRussell2001)
- SDS-PAGEsamplebuffer,2x(SambrookandRussell2001)
- 100mMTris-HCl(pH6.8)
- 4%(w/v)SDS(electrophoresisgrade)
- 0.2%(w/v)bromophenolblue
- 20%(v/v)glycerol
- 200mMdithiothreitol(DTT)orβ-mercaptoethanol
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|
| METHOD |
- Inoculateonecolonyofeachbacterialstrainexpressingeachconstruct(GSTalone,GSTfusionproteins)intoindividual5-mlaliquotsofLBbrothcontainingappropriateantibioticselection.Growovernightat37°Cwithshaking.AlthoughvariationsofGSTfusionproteinexpressionvectorsareavailable,themostcommonlyusedversionsincludethesequenceencodingtheGSTmoietyfollowedbyamultiplecloningsite;anIPTG-induciblepromoter;theampicillin-resistancegene;thelacIgeneforexpressioncontrol;andabacterialoriginofreplication.Manybacterialstrainscanbeused,includingthosecommonlyusedforcloning.Alternatively,protease-deficientstrainssuchasBL21havebeencommonlyusedforexpressionofrecombinantproteins.
- Inoculate1literofLBcontainingantibioticselectionwiththe5-mlovernightculturefromstep1.
- Growat37°CwithshakingtoanOD600of0.5-1.0(shouldtake3-6hr).
- InduceexpressionoftheproteinbyaddingIPTGtoafinalconcentrationof0.1mM.DifferentproteinsareoptimallyproducedusingdifferentconcentrationsofIPTG.Ifproteinexpressionisproblematic,firsttitratetheamountofIPTGaddedtodeterminetheoptimalconditionsforproteininduction.
- Incubateforanadditional3hrat37°Cwithshaking.
- Centrifugethebacterialcultureat3500gfor20minat4°C.
- Discardthesupernatant.Atthispoint,pelletscanbestoredfrozenat-20°Cifnecessary.
- ResUSPendthepelletin20mloflysisbuffersupplementedwithproteaseinhibitors.
- Sonicatethebacterialsuspensiononice,inshort10-secburstsalternatedwith10secrestingonice.Threecyclesofsonicationareusuallysufficient.Oversonicationcanresultindegradationanddenaturationofthefusionprotein.Itcanalsoresultincontaminationwithbacterialhostproteins(thiscanbedetectedafterelutionofthepreparationonanSDS-PAGEgel,andstainingwithCoomassie).Iftheseproblemsoccurinthepreparationoftheproteinofinterest,titrationofthetimeofsonicationrequiredtoreleasetheproteinisuseful.
- Centrifugethelysateat12,000gfor15minat4°Catthisstage.
- Transferthesupernatanttoafreshtube.
- Add5mlofa50:50slurryofglutathione-Sepharosebeadsinlysisbuffer.Commerciallyavailableglutathione-Sepharosebeadsareoftenprovidedinasolutioncontainingalcoholsorotheringredients.Priortouse,theseresinsshouldbewashedwithlysisbufferandstoredasa50:50(v/v)slurryat4°C.
- Incubatefor30minat4°C,rotatingthetubeendoverendtoensuremixing.
- Centrifugeat750gfor1minat4°Ctopelletthebeads.Removethesupernatant.
- Washthebeadsin5mlofice-coldPBSsupplementedwithproteaseinhibitors.
- Centrifugeat750gfor1minat4°Ctopelletthebeads.Removethesupernatant.
- Add5mlofice-coldPBSsupplementedwithproteaseinhibitors.Resuspendthebeadsbygentlemixing.
- Centrifugeagainat750gfor1minat4°Ctopelletthebeads.Removethesupernatant.Thefusionproteincanbestoredonthebeadsat4°Catthisstage.ThisisappropriateiftheproteinistobelabeledorusedinaGSTpull-downexperiment.
- Add5mlofice-coldPBSsupplementedwithproteaseinhibitors.Resuspendthebeadswithgentlemixing.
- Pourtheslurryintoacommerciallyavailabledisposablechromatographycolumn.
- AllowthePBStorunoutofthecolumn.Washwith5mlofice-coldPBSsupplementedwithproteaseinhibitors.
- Whilethecolumnisflowing,preparearackof10microcentrifugetubeslabeled1-10.
- Elutethefusionproteinbyadding5mlofcold(0-4°C)20mMreducedglutathionein50mMTris(pH8.0).
- Collect~0.5-mlfractionsoftheeluateineachmicrocentrifugetube.
- Storetheeluateat4°Cpriortouse.ThecolumncanbestoredinPBSat4°Cproperlysealedtopreventdesiccationandcontamination.Theelutedproteinsareinasolutioncontaining20mMglutathione.Inmostinstances,itisoptimaltoremovetheglutathione.Thiscanbeaccomplishedbydialysisagainstthebufferthatismostcompatiblewiththeassayinwhichtheproteinwillbeutilized.Alternatively,acommerciallypreparedconcentrationbufferexchangeunit(withalowMWcutoff)canbeusedforbufferexchange.
- Performaproteinassayontheelutedfractions.Theresultsoftheproteinassaywillindicatewhichoftheeluatetubescontainsthefusionprotein.RunthesamplesfromtheeluatescontainingproteinonanSDS-polyacrylamidegelandstainwithCoomassiebluedye.TheGSTmoietyis26kD;therefore,add26kDtothepredictedmolecularweightofyourfusionproteintodeterminetheanticipatedmolecularweight.Atthispoint,therecombinantproteincanbestored.Themethodofstoragemustbedeterminedempirically.Forexample,aproteintobeusedsubsequentlyinanenzymaticassaymayrequirespecifichandlingascomparedtoaproteintobeusedinaprotein-proteininteractionstudy.Mostproteinscanbestoredforshortperiodsoftimeat4°C.Ingeneral,freeze-thawcyclesshouldbeavoided.Fortheassaysdescribedbelow,itisimportanttorunaSDS-polyacrylamidegeltochecktheintegrityoftheproteinafterprolongedstorage.
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| Troubleshooting |
- LowYield.Iftheyieldislowattheendofthepurification,repeatproteinpurificationandcomparethelevelsofthefusionproteinpresentatthedifferentstagesofpreparation.Removealiquotsfromthepreparationatthestepsdetailedbelow.CombinethesampleswithSDSsamplebufferandanalyzethemonaSDS-polyacrylamidegel(stainwithCoomassieblue).Volumesarebasedonastartingvolumeofaliterofculture;adjustaccordingly.-15µlofuninducedculturefromstep3(priortoadditionofIPTG)-15µlofinducedculturefromstep5-0.2%ofsupernatantatstep9(totalcelllysate):40µl-0.2%ofsupernatantatstep11(solublelysate):40µl-0.2%ofsupernatantatstep14(lysateafterincubationwithbeads):40µl-0.2%ofaliquotofaneluatefraction(step26):1µl-2%ofaliquotofaneluatefraction(step26):10µlTheGSTmoietyadds~26kDmolecularmass.Ifproteindegradationisoccurring,theMWofthemajorityrecoveredspeciesmaybesignificantlylessthanthepredictedMW;runthegelaccordingly.Resultsfromthisgelwillshow:
- Theyield,andtheintegrityoftheprotein(aliquotsfromstep26).
- Whethertheproteinwasinduced(compareuninducedandinducedcultures,aliquotsfromstep3andstep5).
- Whethertheproteinissoluble(comparealiquotsfromstep9andstep11).
- Whetherasubstantialportionofthefusionproteinboundtothebeads(comparealiquotsfromstep14andstep11).
- FailuretoInduceProteinExpression.Todeterminewhethertheinductionconditionsareworking,prepareacultureofGSTinparallelwiththeGSTfusionprotein.IfGSTisproduced,butthefusionproteinisnot,optimizationofinductionofthefusionproteinisnecessary.GrowthconditionsthatcanbevariedtoaddressthisproblemincludetitratingtheamountofIPTGadded,alteringtheOD600atwhichtheIPTGisadded,loweringthetemperatureofbacterialgrowth,andinducingforlongerorshortertimes.
- DegradationoftheProtein.Theproblemofproteindegradationcanbeaddressedbyusingprotease-deficient(lon-)bacterialstrains.Therearemanycommerciallyavailablebacterialstrainsdesignedforproteinexpressionwithappropriategeneticbackgroundstominimizeproteindegradation(e.g.,BL21).Degradationcanalsoresultfromoversonication.Determinetheoptimalsonicationtimefortheproteinofinterestbyperformingsmall-batchpreparationsandtestingdifferentsonicationtimes.
- Insolubility.Insolubilitycanbeamelioratedbyinducingexpressionatlowertemperatures(30°Corless)andforlongertimes.Theuseofdifferentdetergentsduringlysiscanalsohelp(anexcellentreferenceforthisproblemisFrangoniandNeel1993).Anotherwaytoavoidthisdifficultyistoisolatetheinsolubleproteinandsubsequentlyrefoldit(Volkeletal.1998;Coxetal.1999),butthisisnotalwayspossible.Itmaybethatthefusionproteincontainsdomainsthatpromoteinsolubilityoraggregation(lipid-bindingdomains,forexample).Alteringthefusionprotein,wherepossible,toexcludethesedomainscanimprovesolubility.However,iftheproteincannotbemodified,thesolutionmaybetotransfertheproteinofinteresttoaHis-taggedvector(whichallowsaffinitypurificationofdenaturedproteins).
- FailuretoBindtheGlutathione-Sepharose.Excessivesonicationcanofteninterferewithglutathione-Sepharosebinding.Toaddressthispossibility,decreasethetimeandintensityofsonication.AdditionofDTT,toafinalconcentrationof5mM,priortolysiscanalsoincreasethebindingofsomefusionproteins.
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| REFERENCES |
| Cox,G.N.,PrattD.,McDermottM.J.andvanderSliceR.W.1999.RefoldingandcharacterizationofrecombinanthumansolubleCTLA-4expressedinE.coli.ProteinExpr.Purif.17:26-32.FrangoniJ.V.andNeelB.G.1993.Solubilizationandpurificationofenzymaticallyactiveglutathione-S-transferase(GEX)fusionproteins.Anal.Biochem.210:179-187.SambrookJ.andRussellD.W.2001.MolecularCloning:ALaboratoryManualThirdEdition.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYorkVolkelD.,BlankenfeldtW.,andSchomburgD.1998.Large-scaleproduction,purificationandrefoldingofthefull-lengthcellularprionproteinfromSyriangoldenhamsterinE.coliusingglutathioneS-transferase-fusionsystem.Eur.J.Biochem.251:462-471. |
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| Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:Protein:ProteinInteractions,SecondEdition:AMolecularCloningManual,editedbyEricaA.GolemisandPeterD.Adams,©2005byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.89. |
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| Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission. |
