GST融合蛋白純化方法演示教學(xué)

1、Good is good, but better carries it.精益求精，善益求善。GST融合蛋白純化方法GST融合蛋白純化方法PurificationofGSTFusedProteinsAbstract:ManypeoplehaveventedoutfrustrationoverinsolubleGST-fusedproteins.ThisisaprotocolforenzymaticallyactivesolubleGST-fusedproteins.AllGST-fusedproteinsarerenderedsolublewiththistechniquethoughenzym

2、eactivitiycanrangefrom30-90%.MaterialsandReagentsSTEBuffer10mMTris-HCl,pH8.01mMEDTA150mMNaClLysozymesolution10mg/mlinwater(makefresh)PBSElutionBuffer50mMTris.Cl,pH9.020mMGSH10%SarkosylinSTEBuffer10%TritonX-100inSTEBuffer1MDTT100mMIPTGProcedureDay1Setupanovernightculturein50ml2XTYwith150mg/mlofampici

3、llin.Day2Seed5mlofovernightcultureto500ml2XTYwith150mg/mlofampicillin.Growat37oCtoanA600of0.6to0.8.Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.LowerIPTGconcentrationsandlowergrowingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.Pelletcellsbycentrifuging

4、at3000g,4oCfor10min.Decantmediaandresuspendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4oCfor10min.DecantPBS.Thisisaconvenientpointtostopandtostorepelletsat-80oC.Elsecontinuetolysecells.Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.Resuspendpelletin10mlofi

5、cecoldSTEBuffer.Add100mloffreshlypreparedlyozymesolution,incubateonicefor15min.Justbeforesonication,add100mlof1MDTTand1.4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthep

6、ellet.Add4mlof10%TritonX-100andtopupwithSTEBufferto20ml.TheeffectiveconcentrationofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1hrwithagitation.Topreparethe50%slurry,sh

7、akeupthemediaandpipette2mltoa50mltube.Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.Washthebeadswith3X50mlofPBS.Finallyresuspendin5mlofPBS.Pourtoadispo-column.Washthe50-mlconicaltubewithanadditional5mlo

8、fPBS.Poolwiththefirst5mlinthedispo-column.Towash,usethesamecentrifugationtechniqueforpreparingthebeads.Whentransferringbeadstocolumn,donotpipettebutpour.Thebeadstendtosticktopipettetips.Ifdesired,elutewith10 x1mlfractionsofElutionBuffer.DeterminedesiredfractionswithSDSPAGE親和層析實(shí)驗(yàn)技術(shù)方法INTRODUCTIONThisp

9、rotocoldescribesamethodforremovingantibodiesthatreactwithbacteriallyencodedproteinsbypassingacrudepreparationofimmunoglobulinsthroughacolumncontainingimmobilizedbacterialproteins.MATERIALSReagentsE.colistrainusedashostforpreparationofexpressionlibraryAntibodypreparationthatistobeusedforscreeningThis

10、protocolworksbestwhenusinganIgGfraction,preparedbychromatographyoftheantiserumonproteinA-Sepharose.Celllysisbuffer0.1Msodiumborate(pH8.0)1MNaClSterilizethecelllysisbufferusinga0.45-mfilter,andstoreatroomtemperature.Approximately100mlofcelllysisbufferisrequiredper1literofbacterialculture.Growthmedium

11、OneliterofgrowthmediumappropriatefortheE.colistrainofchoiceisrequired.LysozymeDissolvesolidlysozymeataconcentrationof10mg/mlin10mMTris-Cl(pH8.0)immediatelybeforeuse.MakesurethatthepHoftheTrissolutionis8.0beforedissolvingtheprotein.LysozymewillnotworkefficientlyifthepHofthesolutionislessthan8.0.Useam

12、olecularbiologygradeoflysozyme.Addsolidlysozymetoassistlysisofbacterialcells.NaOH(1N)Thepreparationof10NNaOHinvolvesahighlyexothermicreaction,whichcancausebreakageofglasscontainers.Preparethissolutionwithextremecareinplasticbeakers.To800mlofH2O,slowlyadd400gofNaOHpellets,stirringcontinuously.Asanadd

13、edprecaution,placethebeakeronice.Whenthepelletshavedissolvedcompletely,adjustthevolumeto1literwithH2O.Storethesolutioninaplasticcontaineratroomtemperature.Sterilizationisnotnecessary.PancreaticDNaseI1mg/mlPancreaticDNaseI50mMNaCl10mMTris-Cl(pH7.5)1mMMgCl2Dissolve2mgofcrudepancreaticDNaseI(Sigmaorequ

14、ivalent)in1mlof50mMNaCl,Tris-Cl(pH7.5),1mMMgCl2.WhentheDNaseIisdissolved,add1mlofglyceroltothesolutionandmixbygentlyinvertingtheclosedtubeseveraltimes.Takecaretoavoidcreatingbubblesandfoam.Storethesolutioninaliquotsof-20C.AddsolidDNaseItothebacterialcelllysatetodigestchromosomalDNA.Tris-bufferedSali

15、ne(TBS)Dissolve8gofNaCl,0.2gofKCl,and3gofTrisbasein800mlofdistilledH2O.Add0.015gofphenolredandadjustthepHto7.4withHCl.AdddistilledH2Oto1liter.Dispensethesolutionintoaliquotsandsterilizethembyautoclavingfor20minutesat15psi(1.05kg/cm2)onliquidcycle.Storethebufferatroomtemperature.TBScontaining0.2%(w/v

16、)sodiumazideTritonX-100METHODGrowa1-litercultureoftheappropriatestrainofE.coli(e.g.,Y1090hsdR,XL1-Blue,orDH1)tostationaryphase.Recoverthebacteriabycentrifugationat4000g(5000rpminaSorvallGSArotor)for20minutesat4C.Pouroffthemedium,andstandthecentrifugetubesinaninvertedpositiontoallowthelasttracesofmed

17、iumtodrainaway.Resuspendthepelletin100mlofCelllysisbuffer.Add200mgoflysozyme,andincubatethebacterialsuspensionfor20minutesatroomtemperature.Add1mgofpancreaticDNaseIand200lofTritonX-100.Incubatethebacterialsuspensionfor1hourat4C,oruntiltheturbidityclearsandtheviscositydecreases.Centrifugethebacterial

18、lysateat8000g(8200rpminaSorvallSS-34rotor)for20minutesat4C.Carefullydecantthesupernatantintoafreshflask.AdjustthepHofthesupernatantto9.0with1MNaOH.DeterminetheconcentrationofproteininthelysateusingtheLowry,Bradford,orothermethodofmeasurement.Chilltheextractto0C,andthenbindthebacterialproteinstocyano

19、gen-bromide-activatedSepharose4BortoAffi-Gel10accordingtothemanufacturersinstructions.Beforeuse,equilibratetheSepharose4BorAffi-Gel10resincontainingconjugatedE.coliproteinsinTBScontaining0.2%(w/v)sodiumazide.Use1mlofsettledvolumeofresincoupledtoE.coliantigenforeachmilligramofIgGproteintobepurifiedby

20、affinitychromatography.MixtheIgGandthecoupledresinandincubatefor12-18hoursatroomtemperatureonarotatingwheeldevice.Loadtheslurryintoachromatographycolumn.RecovertheantibodybywashingthecolumnwithTBS.Collectfractions(0.2columnvolumeeach)untiltheOD280dropstozero.Poolthefractionscontainingantibody,andsto

21、rethepoolat-20Cuntilitisusedforimmunologicalscreening.REFERENCES1.deWet,J.R.,Fukushima,H.,Dewji,N.N.,Wilcox,E.,OBrien,J.S.,andHelinski,D.R.1984.Chromogenicimmunodetectionofhumanserumalbuminandalpha-L-fucosidaseclonesinahumanhepatomacDNAexpressionlibrary.DNA3:437-447.MedlineAnyoneusingtheproceduresin

22、thisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsre

23、gardingthesematerial,pleaseconsult:MolecularCloning:ALaboratoryManual,ThirdEdition,JosephSambrookandDavidW.Russell,2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.14.28-14.30.GST融合蛋白純化方法1目的片段接入pGEX載體；2涂板，挑單克隆，搖菌至OD6001.0，加入IPTG（終濃度1mM）誘導(dǎo)68h；3收菌，每升菌液約以50mLPBS重懸，加入1%TritonX-100（v/v），1

24、-巰基乙醇（v/v），PMSF（終濃度1mM）；以下步驟均在冰上操作：4超聲破碎菌體，15000g，10min離心取上清，在上清中加入適量GST-beads，輕輕晃動令其吸附蛋白1h；52000g，3min離心棄上清；6加入至少10倍體積PBS，輕搖至beads懸浮于溶液中，2000g，3min離心棄上清；7重復(fù)步驟6兩次；8加入1mLGSTElutionBuffer，輕搖10min；92000g，3min離心，收集上清；10重復(fù)步驟8-9至少兩次；11SDS電泳檢測蛋白純度，Bradford法檢測蛋白濃度；12將蛋白置于-20保存。P.S.大量提取前應(yīng)取少量菌液，改變IPTG濃度，誘導(dǎo)溫度，

25、誘導(dǎo)時間等，以確定蛋白表達(dá)的最適條件。GSTfusionproteinpurificationGST融合蛋白純化1)grow20mlcellsO/N37dilute50XintoprewarmedLB,growto0.6ODorabout1hr.Inducew/2mMIPTG(238mg/0.5l),grow3hr,spin6kGS310,freezeat702)extractcells(from500ml)in25mls.HeintzBufferplustriton(HBT)bygentlepipetteresuspendingonicecirca10afterthawing.3)trans

26、ferto50mlconicalss34fliptoptubes.4)add10mglysozymepowdertothe50ml(cellsfrom1liternowin150mltube),digest15onice.5)sonicatewithlargeprobe180%power,freezeinlN,thawat37sonicateagainonice,solutionshouldbecomeviscous.6)add1mgDNAseandRNAse,incubateonice15.7)spin7.5krpm4ss3410.8)transfersupernatantstoconica

27、lscrewcaps,freezeinlN2,maystore.9)Batchadsorbw/4ml50%slurryGT-Sepharose(PL17-0756-01),1hr,4spin2,4konbench.10)aspirate,resuspendin25mlHBT,spin,repeat.11)pourslurryintocolumn(Econo1.7x20),elutetotop,thenwith20columnvols.ofHB-T.12)eluteproteininminimalvolume(5-10ml)HB5mMGT(SigmaG4251,1.5mg/ml).13)lN2f

28、reezeas100mlaliquotsforGS.HB1literFinalStockml/l25mMHEPES,pH7.91M501mMEDTA,pH8.00.5M220%glycerolstock2001mMMgCl21M160mMKCl2M301%Tritonstock10addbeforeuse0.5mMDTT1M0.50.5mMPMSF0.5mM105g/mlLeupeptin5mg/mldilutebeforeuse5mg/mlantipaincheckpH!GUSHISTOCHEMICALSTAINING1)determinenumberofslidesneeded,multi

29、plyby0.75ml2)makeuprequiredvolofstain:for10ml45mgX-Gus50lnndimethylformamide,dissolve10ml50mMNaPO4pH73)sectionsbestcutwithvibratingknifeforsectionsw/chlorophyll,putincell-wellsw/500lstainforsectionsw/outchlorophyll,putdirectlyonslidesw/stain4)inco/n37inhumiditychamber5)asp,inc10inFAA:for200ml410mlfo

30、rmaldehyde10mlHAc75mlEtOHH2Ovol6)inc250%EtOH7)inc2100%EtOH8)inc1H2OPurificationofGSTfusionproteinsinE.coliGST融合蛋白純化，方法一MakingGSTfusionproteins:(07/19/03)ver.1Growup5mlLBwithAmpo/n.Addto45mlLBwithAmp37oshake2.5-3hrs,tillOD6000.4-0.8Putbottlesinroomtemperaturewaterfor10mintocooldown.Add100l0.2MIPTGto0

31、.4mMfinalShake302hrPelletbacteria,decantsup,inverttodrainResuspendin1mlNETN0.2mMPMSF/50mlLBPMSF,stock10mMNETN:20mMTris-Cl(pH8)100mMNaCl1mMEDTA0.5%NP40storeat4VortextomixwellSonicateatscale5for15sec.Keeponice.For10mlCorningtubes,usescale7Spin4,5minTransfersupernatanttoanewtube.Toeachlysate,add60l50%G

32、lutathione-Sepharose4BPepette400lSepharosestock(75%)Spin1000rpm5min,discardsupernantantWash3x300lNETNResuspendin300lNETNtoget50%beadsMixincoldroomfor2hours,slowlywhirlPelletbeadsbybriefcentrifugation,carefullydiscardsupernatantWash3x1mlNETN/PMSFWash2x1mlElutionBuffer(50mMHepes,pH7.9,40mMKCl,1mMEDTA1

33、mMDTT)Eluteproteinsbymixbeadswith60leachElutionbuffer5mMGlutathione,(for10mM,use3.07mg/ml)1mMDTTSlowlyswirlatRT1hrQuickspintopellet,transfersupernatanttoanewtubeRe-elutewith60leachNETN5mMGlutathione1mMDTTSlowlyswirlatRT30minQuickspin,combinesupernatant,spinandtransfersupernatanttwicetoavoidanyresidu

34、albeads.totalis120lnow.Dialyzevs50%glycerol/10mMHepes,pH7.5/40mMKCl/1mMEDTA/1mMDTT/1mMPMSFincoldroomfor2hroro/n,storeat-20ProteinscanalsobeconcentratedinaCentriprep-30concentrator.TheporesizeofthemembraneintheCentriprep-30allowsglutathionetopassintotheaqueouscompartment.PBScanbeaddedtotheproteinconc

35、entrateandtheconcentrationprocedurecanberepeated.Thinkingaliquotandsaveat-80Run12%SDSPurificationofGSTfusionproteinsinE.coliGST融合蛋白純化，方法二GSTProteinPrep.Ver.21)Grow50mlofcultureinLBorTBantibiotico/nat37shaker.2)DilutecultureinLBorTBantibiotic1:103)Grow3hrsat37.4)Induceculturebyadding0.4mMIPTGfinalcon

36、centration.(For50mlfinalcultureadd20lof1MIPTG).5)Growat25for1hr.6)Spin5minat5K7)WashpelletwithhalfvolumeofcoldH2O.(For50mlcultureuse25mlH2O.)8)Washbacteriaagain.9)Resuspendin1mlofresuspensionbufferper50mlofculture.ResuspensionBuffer-NETNproteaseinhibitors20mMTrispH8.0100mMNaCl1mMEDTA0.5%NP-40orTrito

37、n-X1g/mlAproteinin1g/mlPMSF1g/mlBenzaminideNote:Timhas100 xstockofproteaseinhibitors.10)Sonicatefor2xfor10secondsincoldroom.11)Pelletdebrisbyspinningat4(Easiestwayistoputsamplesinmultipleeppendorfsandspinincoldroomatmaxfor2min.BindingofFusionProteintoBeads1)Pipette400lofGST-SepharoseintoEppendorf2)S

38、pinbeads15sec8,000RPM3)Washbeads2xwith4NETN4)Resuspendpelletin320lofNETN(Finalvolumewillbe550l)andputinto4eppendorfs.5)Add300lofresuspensionbufferand75lofE.Coli.GSTLysatetoeachtube.6)Incubatefor30minwhilerockingincoldroom.7)Spin15secondsat8,000rpm.8)Wash3xwith600lofresuspensionbuffer9)Removesupernat

39、antandresuspendinappropriatevolumeresuspensionbuffer.(YoucanaddthisdirectlytoSDSloadbufferforrunningonagel.)PurificationofGSTfusionproteinsinE.coliGST融合蛋白純化，方法三，純化小量SmallscalefusionproteinpreparationGrow5mlcultureo/ninTBwithamp.Addo/ncultureto50mlofTBampandgrowfor3hoursin37shaker.Induceculturebyaddi

40、ng20lof1MIPTG(final0.4mM)andtransferto25shakerfor1hour.PelletBacteria10minat3KResuspendbacteriain1mlofNETNproteaseinhibitors.Sonicate2xfor5-10secondseachtime.Spinoutcelldebrisbyspinningincoldmicrofuge5min.atmax.Removesupernatantandstoreat-70.BindingfusionproteinlysatetobeadsRemove400lofGSTbeadsintoe

41、ppendorfandspin15secat8k.Removesupernatantandwash2xwithNETN.Resuspendbeadsin320lofNETN(550lfinalvolume)Aliquot50lofbeadsandaddupto200lofGSTlysate.If200lbringfinalvol.upto200lwithNETN.Incubateat4withrockingfor30min.Wash3xwithNETNandaddlysatecontainingproteinofinterest.Incubate1-2hrat4withrocking.Wash

42、3xwithappropriatesaltbuffer.Boilproteinsoffin2xSBforwesternblotting.NETN2xSB20mMTrispH8.0125mMTrispH6.8100mMNaCl20%Glycerol1mMEDTA4.1%SDS0.5%NP-402%BME0.005%BromphenolBlueGSTPurificationLargeScaleProtocolResuspend500mlpelletin10mlofNETNandmixwell(Keepat4)Combinetwopellets(20ml)andtransfer20mlofresus

43、pensionto50mlconicalSonicate2xwith15secpulsesSpinfor20at10kinSorvallat4.Transfersupernatantintonew50mltubes.Add1mlofwashedbeadsrockfor30minatRTPoursampleintocolumnanddrainSaveflowthroughandrepeatpreviousstep2xWashbeadswith10mlof1xPBS3xAdd500lofElutionbufferandrockfor10minatRTDrainandcollect500loffra

44、ctionRepeat2xmoreElutionBuffer(5ml)NETN20mMTrispH8.010mMglutathione15.6mg100mMNaCl50mMTrispH8.0125l1mMEDTAH2O4.9ml0.5%NP-40PurificationofGSTfusionproteinsinE.coliGST融合蛋白純化（方法四）篩選表達(dá)ScreenofGST-FusionProteinExpression(pGEXsystembyAmersham:forcheckclonesforexpressionofthedesiredfusionproteinpriortolarg

45、e-scalepurification)PickseveralcoloniesofE.colitransformedwiththepGEXrecombinantsintoseparatetubescontaining2mlof2xYTAmedium.-Note:Forcomparison,itisadvisabletoinoculateacontroltubewithbacteriatransformedwiththeparentalpGEXplasmid.GrowliquidculturestoanA600of0.6-0.8(35h)withvigorousagitationat2037.I

46、ncubatefusionproteinexpressionbyadding2lof100mMIPTG(finalconcentration0.1mM).Continueincubationforanadditional12hTransfer1.5mloftheliquidculturestolabeled1.5mlmicrocentrifugetubes.Centrifugeinamicrocentrifugefor5secanddiscardthesupernatant.Resuspendeachpelletin300lofice-cold1xPBS,remove10loftheseres

47、uspendcellsintolabeledtubes(forlateruseinSDSanalysis).-Note:Exceptwherenoted,keepallsamplesandtubesonice.Lysethecellsusingthesonicatorequippedwithanappropriateprobe.-Note:Lysisiscompletewhenthecloudycellsuspensionbecomestranslucent.Thefrequencyandintensityofsonicationshouldbeadjustedsuchthatcomplete

48、lysisoccursin10sec,withoutfrothing(itmaydenatureproteins).-Note:CrudesonicatescanbescreenedfortherelativelevelofexpressionofGSTfugionproteinsusingtheGSTsubstratesCDNB(1-chloro-2,4-dinitrobenzene).Centrifugeinamicrocentrifugefor5mintoremoveinsolublematerials.Savea10laliquotsoftheinsolublematerialfora

49、nalysisbySDS.Transferthesupernatantstofreshtubes,Add20lofa50%slurryofGlutathioneSepharose4B(preparedasdescribedabove)toeachsupernatantandmixgentlyfor5minatr.t.Add100lof1xPBS,vortexbriefly,andcentrifugefor5sectosedimenttheSepharosebeads.Discardthesupernatant,repeatthis1xPBSwashtwiceElutethefusionprot

50、einbyadding10lofGlutathioneElutionBuffer.SuspendtheSepharosebeadsandincubatefor5minatr.t.Centrifugeinamicrocentrifugefor5mintosedimenttheSepharosebeads,thentransferthesupernatanttofreshtubes.*Glutathioneelutionbuffer:10mMreducedglutathionein50mMTris-HCl(pH8.0).Dispensein1-10mlaliquotsandstoreat20unt

51、ilneeded.Avoidmorethanfivefreeze/thawcycles.PreparationofGlutathioneSepharose4B(forbulkmatrixforbatchpurification)GentlyshakethebottleofGlutathioneSepharose4Btoresuspendthematrix.Useapipettoremovesufficientslurryforuseandtransfertoanappropriatecontainer/tube.(GlutathioneSepharose4Bassuppliesisapprox

52、imatelya75%slurry.Thefollowingprocedureresultsina50%slurry;Basedonthebedvolumerequirement,dispense1.33mloftheoriginalGlutathioneSepharose4Bslurrypermlofbedvolumerequired).Sedimentthematrixbycentrifugationat500 xgfor5min,carefullydecantthesupernatant.WashtheGlutathioneSepharose4Bbyadding10molofcold(4

53、)1xPBSper1.33mloftheoriginallyslurryofGlutathioneSepharose4Bdispensed,Inverttomix.-Note:GlutathioneSepharose4Bmustbethoroughlywashedwith1xPBStoremovethe20%ethanolstoragesolution.Residualethanolmayinterferewithsubsequentprocedures.Sedimentthematrixbycentrifugationat500 xgfor5min.Decantthesupernatant.

54、Foreach1.33mloftheoriginalslurryofGlutathioneSepharose4B,and1mlof1xPBS.Thisresultsina50%slurry.Mixwellpriortosubsequentpipettingsteps.-Note:GlutathioneSepharose4Bequilibratedwith1xPBSmaybestoredat4forupto1month.BulkpurificationofproteinexpressedinE.coliPickseveralcoloniesofE.colitransformedwiththepGEXrecombinantsintoseparatetubescontaining2mlof2xYTAmedium.Growo/nat2037.Add2mlcultureinto100ml2xYTAmedium.GrowliquidculturestoanA600of0.6-0.8(about2h)withvigorousagitationat2037.Incubatefusionproteinexpressionbya