GST融合蛋白純化方法

1、GST融合蛋白純化方法PurificationofGSTFusedProteinsAbstract:ManypeoplehaveventedoutfrustrationoverinsolubleGST-fusedproteins.ThisisaprotocolforenzymaticallyactivesolubleGST-fusedproteins.AllGST-fusedproteinsarerenderedsolublewiththistechniquethoughenzymeactivitiycanrangefrom30-90%.MaterialsandReagentsSTEBuffe

2、r10mMTris-HCl,pH8.01mMEDTA150mMNaClLysozymesolution10mg/mlinwater(makefresh)1. PBSElutionBuffer50mMTris.Cl,pH9.020mMGSH2. 10%SarkosylinSTEBuffer3. 10%TritonX-100inSTEBuffer4. 1MDTT100mMIPTGProcedureDay11. Setupanovernightculturein50ml2XTYwith150mg/mlofampicillin.Day21. Seed5mlofovernightcultureto500

3、ml2XTYwith150mg/mlofampicillin.2. Growat37°CtoanA600of0.6to0.8.3. Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.LowerIPTGconcentrationsandlowergrowingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.4. Pelletcellsbycentrifugingat3000g,4oCfor10min.Decan

4、tmediaandresuspendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4oCfor10min.DecantPBS.5. Thisisaconvenientpointtostopandtostorepelletsat-80oC.Elsecontinuetolysecells.6. Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.7. Resuspendpelletin10mloficecoldSTEBuffer.

5、8. Add100mloffreshlypreparedlyozymesolution,incubateonicefor15min.Justbeforesonication,add100mlof1MDTTand1.4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.9. Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthepellet.Add4

6、mlof10%TritonX-100andtopupwithSTEBufferto20ml.TheeffectiveconcentrationofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.10. Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1hrwithagitation.Topreparethe50%slurry,shakeupt

7、hemediaandpipette2mltoa50mltube.Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.11. Washthebeadswith3X50mlofPBS.Finallyresuspendin5mlofPBS.Pourtoadispo-column.Washthe50-mlconicaltubewithanadditional5mlofP

8、BS.Poolwiththefirst5mlinthedispo-column.Towash,usethesamecentrifugationtechniqueforpreparingthebeads.Whentransferringbeadstocolumn,donotpipettebutpour.Thebeadstendtosticktopipettetips.12. Ifdesired,elutewith10x1mlfractionsofElutionBuffer.DeterminedesiredfractionswithSDSPAGE親和層析實驗技術(shù)方法INTRODUCTIONThis

9、protocoldescribesamethodforremovingantibodiesthatreactwithbacteriallyencodedproteinsbypassingacrudepreparationofimmunoglobulinsthroughacolumncontainingimmobilizedbacterialproteins.MATERIALS ReagentsE.colistrainusedashostforpreparationofexpressionlibraryAntibodypreparationthatistobeusedforscreeningTh

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

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

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

13、sanaddedprecaution,placethebeakeronice.Whenthepelletshavedissolvedcompletely,adjustthevolumeto1literwithH2O.Storethesolutioninaplasticcontaineratroomtemperature.Sterilizationisnotnecessary. PancreaticDNaseIo1mg/mlPancreaticDNaseIo50mMNaClo10mMTris-Cl(pH7.5)o1mMMgCl2Dissolve2mgofcrudepancreaticDNaseI

14、(Sigmaorequivalent)in1mlof50mMNaCl,Tris-Cl(pH7.5),1mMMgCl2.WhentheDNaseIisdissolved,add1mlofglyceroltothesolutionandmixbygently精品文檔invertingtheclosedtubeseveraltimes.Takecaretoavoidcreatingbubblesandfoam.Storethesolutioninaliquotsof-20°C.AddsolidDNaseItothebacterialcelllysatetodigestchromosomal

15、DNA.Tris-bufferedSaline(TBS)Dissolve8gofNaCl,0.2gofKCl,and3gofTrisbasein800mlofdistilledH2O.Add0.015gofphenolredandadjustthepHto7.4withHCl.AdddistilledH2Oto1liter.Dispensethesolutionintoaliquotsandsterilizethembyautoclavingfor20minutesat15psi(1.05kg/cm2)onliquidcycle.Storethebufferatroomtemperature.

16、 TBScontaining0.2%(w/v)sodiumazideTritonX-100METHODGrowa1-litercultureoftheappropriatestrainofE.coli(e.g.,Y1090hsdR,XL1-Blue,orDH1)tostationaryphase. Recoverthebacteriabycentrifugationat4000g(5000rpminaSorvallGSArotor)for20minutesat4°C. Pouroffthemedium,andstandthecentrifugetubesinaninvertedpos

17、itiontoallowthelasttracesofmediumtodrainaway. Resuspendthepelletin100mlofCelllysisbuffer. Add200mgoflysozyme,andincubatethebacterialsuspensionfor20minutesatroomtemperature. Add1mgofpancreaticDNaseIand200lofTritonX-100. Incubatethebacterialsuspensionfor1hourat4C,oruntiltheturbidityclearsandtheviscosi

18、tydecreases. Centrifugethebacteriallysateat8000g(8200rpminaSorvallSS-34rotor)for20minutesat4C.Carefullydecantthesupernatantintoafreshflask.+AdjustthepHofthesupernatantto9.0with1MNaOH. DeterminetheconcentrationofproteininthelysateusingtheLowry,Bradford,orothermethodofmeasurement. Chilltheextractto0&#

19、176;C,andthenbindthebacterialproteinstocyanogen-bromide-activatedSepharose4BortoAffi-Gel10accordingtothemanufacturer'sinstructions. Beforeuse,equilibratetheSepharose4BorAffi-Gel10resincontainingconjugatedE.coliproteinsinTBScontaining0.2%(w/v)sodiumazide. Use1mlofsettledvolumeofresincoupledtoE.co

20、liantigenforeachmilligramofIgGproteintobepurifiedbyaffinitychromatography.MixtheIgGandthecoupledresinandincubatefor12-18hoursatroomtemperatureonarotatingwheeldevice.Loadtheslurryintoachromatographycolumn.RecovertheantibodybywashingthecolumnwithTBS.Collectfractions(0.2columnvolumeeach)untiltheOD280dr

21、opstozero.Poolthefractionscontainingantibody,andstorethepoolat-20Cuntilitisusedforimmunologicalscreening.REFERENCES精品文檔1.deWet,J.R.,Fukushima,H.,Dewji,N.N.,Wilcox,E.,O'Brien,J.S.,andHelinski,D.R.1984.Chromogenicimmunodetectionofhumanserumalbuminandalpha-L-fucosidaseclonesinahumanhepatomacDNAexpr

22、essionlibrary.DNA3:437-447.MedlineAnyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhaz

23、ardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,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體；2涂板，挑單克隆，搖菌至OD60滲1.0，加入

24、IPTG（終濃度1mM）誘導(dǎo)6-8h;3收菌，每升菌液約以50mLPB拆懸，加入1%TritonX-100（v/v）,1%3-疏基乙醇（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；加入1mLGSTElutionBuffer，輕搖10min;2000g,3min離心，收集上清；10重復(fù)步驟8-9至少兩次；11SDS-PAGE電泳檢測蛋

25、白純度，Bradford法檢測蛋白濃度；12將蛋白置于-20C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，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grow20mlcellsO/N37Cdilute50XintoprewarmedLB,growto0.6ODorabout1hr.Inducew/2mMIPTG(238mg/0.5l),grow3hr,spin6kGS310',freezeat70Cextractcells(from500ml)in25mls.HeintzBufferplustri

26、ton(HBT)bygentlepipetteresuspendingonicecirca10'afterthawing.1) transferto50mlconicalss34fliptoptubes.2) add10mglysozymepowdertothe50ml(cellsfrom1liternowin150mltube),digest15'onice.3) sonicatewithlargeprobe1'80%power,freezeinlN,thawat37Csonicateagainonice,solutionshouldbecomeviscous.4)

27、add1mgDNAseandRNAse,incubateonice15'.5) spin7.5krpm4Css3410'.6) transfersupernatantstoconicalscrewcaps,freezeinlN2,maystore.7) Batchadsorbw/4ml50%slurryGT-Sepharose(PL17-0756-01),1hr,4Cspin2',4konbench.8) aspirate,resuspendin25mlHBT,spin,repeat.9) pourslurryintocolumn(Econo1.7x20),elutet

28、otop,thenwith20columnvols.ofHB-T.10) eluteproteininminimalvolume(5-10ml)HB5mMGT(SigmaG4251,1.5mg/ml).11) lN2freezeas100mlaliquotsforGS.精品文檔HB1literFinalStockml/l25mMHEPES,pH7.91M501mMEDTA,pH8.00.5M220%glycerolstock2001mMMgCl2IM160mMKCl2M301%Tritonstock10addbeforeuse0.5mMDTT1M0.5”0.5mMPMSF0.5mM10&quo

29、t;5g/mlLeupeptin5mg/mldilutebeforeuse5mg/mlantipain""checkpH!GUSHISTOCHEMICALSTAINING1) determinenumberofslidesneeded,multiplyby0.75ml2) makeuprequiredvolofstain:for10ml4C5mgX-Gus50Unndimethylformamide,dissolve10ml50mMNaPO4pH73) sectionsbestcutwithvibratingknifeforsectionsw/chlorophyll,put

30、incell-wellsw/500lstainforsectionsw/outchlorophyll,putdirectlyonslidesw/stain4) inco/n37Cinhumiditychamber5) asp,inc10'inFAA:for200ml4C10mlformaldehyde10mlHAc75mlEtOHH2O>vol6) inc2'50%EtOH7) inc2'100%EtOH8) inc1'H2O精品文檔PurificationofGSTfusionproteinsinE.coliGSTMakingGSTfusionprote

31、ins:(07/19/03)ver.1Growup5mlLBwithAmpo/n.Addto45mlLBwithAmp37oshake2.5-3hrs,tillOD6000.4-0.8Putbottlesinroomtemperaturewaterfor10mintocooldown.Add100卬l0.2MIPTGto0.4mMfinalShake30C2hrPelletbacteria,decantsup,inverttodrainResuspendin1mlNETN0.2mMPMSF/50mlLBPMSF,stock10mMNETN:20mMTris-Cl(pH8)100mMNaCl1m

32、MEDTA0.5%NP40storeat4CVortextomixwellSonicateatscale5for15sec.Keeponice.For10mlCorningtubes,usescale7Spin4C,5minTransfersupernatanttoanewtube.Toeachlysate,add60l50%GlutathioneSepharose4BPepette400lSepharosestock(75%)Spin1000rpm5min,discardsupernantantWash3x300lNETNResuspendin300lNETNtoget50%beadsMix

33、incoldroomfor2hours,slowlywhirlPelletbeadsbybriefcentrifugation,carefullydiscardsupernatantWash3x1mlNETN/PMSFWash2x1mlElutionBuffer(50mMHepes,pH7.9,40mMKCl,1mMEDTA1mMDTT)Eluteproteinsbymixbeadswith60leachElutionbuffer5mMGlutathione,(for10mM,use3.07mg/ml)1mMDTTSlowlyswirlatRT1hrQuickspintopellet,tran

34、sfersupernatanttoanewtubeRe-elutewith60leachNETN5mMGlutathione1mMDTTSlowlyswirlatRT30minQuickspin,combinesupernatant,spinandtransfersupernatanttwicetoavoidanyresidualbeads.totalis120wnoDialyzevs50%glycerol/10mMHepes,pH7.5/40mMKCl/1mMEDTA/1mMDTT/1mMPMSFincoldroomfor2hroro/n,storeat-20CProteinscanalso

35、beconcentratedinaCentriprep-30concentrator.TheporesizeofthemembraneintheCentriprep-30allowsglutathionetopassintotheaqueouscompartment.PBScanbeaddedtotheproteinconcentrateandtheconcentrationprocedurecanberepeated.Thinkingaliquotandsaveat-80CRun12%SDS-PAGEPurificationofGSTfusionproteinsinE.coliGST融合蛋白

36、純化，方法二GSTProteinPrep.Ver.21) Grow50mlofcultureinLBorTBantibiotico/nat37Cshaker.2) DilutecultureinLBorTBantibiotic1:10lof13) Grow3hrsat37C.4) Induceculturebyadding0.4mMIPTGfinalconcentration.(For50mlfinalcultureadd20IPTG).5) Growat25Cfor1hr.6) Spin5minat5K7) WashpelletwithhalfvolumeofcoldH2O.(For50ml

37、cultureuse25mlH2O.)8) Washbacteriaagain.9) Resuspendin1mlofresuspensionbufferper50mlofculture.ResuspensionBuffer-NETNproteaseinhibitors20mMTrispH8.0100mMNaCl1mMEDTA0.5%NP-40orTriton-X1g/mlAproteinin1卬g/mlPMSF1g/mlBenzaminideNote:Timhas100xstockofproteaseinhibitors.10) Sonicatefor2xfor10secondsincold

38、room.Pelletdebrisbyspinningat4C(Easiestwayistoputsamplesinmultipleeppendorfsandspinincoldroomatmaxfor2min.BindingofFusionProteintoBeads1) Pipette400lofGSSepharoseintoEppendorf2) Spinbeads15sec8,000RPM3) Washbeads2xwith4CNETN4) Resuspendpelletin320lofNETN(Finalvolumewillbe550l)andputinto4eppendorfs.5

39、) Add300lofresuspensionbufferand75lofE.Coli.GSTLysatetoeachtube.6) Incubatefor30minwhilerockingincoldroom.7) Spin15secondsat8,000rpm.8) Wash3xwith600lofreuspensionbufferRemovesupernatantandresuspendinappropriatevolumeresuspensionbuffer.(YoucanaddthisdirectlytoSDSloadbufferforrunningonagel.)Purificat

40、ionofGSTfusionproteinsinE.coliGST融合蛋白純化，方法三，純化小量SmallscalefusionproteinpreparationGrow5mlcultureo/ninTBwithamp.Addo/ncultureto50mlofTBampandgrowfor3hoursin37Cshaker.Induceculturebyadding20lof1MIPTG(final0.4mM)andtransferto25Cshakerfor1hour.PelletBacteria10minat3KResuspendbacteriain1mlofNETNproteasei

41、nhibitors.Sonicate2xfor5-10secondseachtime.Spinoutcelldebrisbyspinningincoldmicrofuge5min.atmax.Removesupernatantandstoreat-70C.BindingfusionproteinlysatetobeadsRemove400lofGSTbeadsintoeppendorfandspin15secat8k.Removesupernatantandwash2xwithNETN.Resuspendbeadsin320lofNETN(550lfinalvolume)Aliquot50lo

42、fbeadsandaddupto200lofGSTlysate.If<200lbringfinalvol.upto200NETN.Incubateat4Cwithrockingfor30min.Wash3xwithNETNandaddlysatecontainingproteinofinterest.Incubate1-2hrat4Cwithrocking.Wash3xwithappropriatesaltbuffer.Boilproteinsoffin2xSBforwesternblotting.精品文檔精品文檔NETN2xSB20mMTrispH8.0125mMTrispH6.810

43、0mMNaCl20%Glycerol1mMEDTA4.1%SDS0.5%NP-402%BME0.005%BromphenolBlueGSTPurificationLargeScaleProtocolResuspend500mlpelletin10mlofNETNandmixwell(Keepat4C)Combinetwopellets(20ml)andtransfer20mlofresuspensionto50mlconicalSonicate2xwith15secpulsesSpinfor20at10kinSorvallat4C.Transfersupernatantintonew50mlt

44、ubes.Add1mlofwashedbeadsrockfor30minatRTPoursampleintocolumnanddrainSaveflowthroughandrepeatpreviousstep2xWashbeadswith10mlof1xPBS3xAdd500lofElutionbufferandrockfor10minatRTDrainandcollect500loffractionRepeat2xmoreElutionBuffer(5ml)NETN20mMTrispH8.010mMglutathione15.6mg100mMNaCl50mMTrispH8.0125出1mME

45、DTAH2O4.9ml0.5%NP-40精品文檔融合蛋白純化（方法四）篩選表達(dá)PurificationofGSTfusionproteinsinE.coliGSTScreenofGST-FusionProteinExpression(pGEXsystembyAmersham:forcheckclonesforexpressionofthedesiredfusionproteinpriortolarge-scalepurification)PickseveralcoloniesofE.colitransformedwiththepGEXrecombinantsintoseparatetubesc

46、ontaining2mlof2xYTAmedium.-Note:Forcomparison,itisadvisabletoinoculateacontroltubewithbacteriatransformedwiththeparentalpGEXplasmid.GrowliquidculturestoanA600of0.6-0.8(35h)withvigorousagitationat2037C.Incubatefusionproteinexpressionbyadding2lof100mMIPTG(finalconcentration0.1mM).Continueincubationfor

47、anadditional12hTransfer1.5mloftheliquidculturestolabeled1.5mlmicrocentrifugetubes.Centrifugeinamicrocentrifugefor5secanddiscardthesupernatant.Resuspendeachpelletin300lof-celd1xPBS,remove10loftheseresuspendcellsintolabeledtubes(forlateruseinSDS-PAGEanalysis).-Note:Exceptwherenoted,keepallsamplesandtu

48、besonice.Lysethecellsusingthesonicatorequippedwithanappropriateprobe.-Note:Lysisiscompletewhenthecloudycellsuspensionbecomestranslucent.Thefrequencyandintensityofsonicationshouldbeadjustedsuchthatcompletelysisoccursin10sec,withoutfrothing(itmaydenatureproteins).-Note:Crudesonicatescanbescreenedforth

49、erelativelevelofexpressionofGSTfugionproteinsusingtheGSTsubstratesCDNB(1-chloro-2,4-dinitrobenzene).Centrifugeinamicrocentrifugefor5mintoremoveinsolublematerials.Savea10laliquotsoftheinsolublematerialforanalysisbySDS-PAGE.Transferthesupernatantstofreshtubes,Add20lofa50%slurryofGlutathioneSepharose4B

50、(preparedasdescribedabove)toeachsupernatantandmixgentlyfor5minatr.t.Add100lof1xPBS,vortexbriefly,andcentrifugefor5sectosedimenttheSepharosebeads.Discardthesupernatant,repeatthis1xPBSwashtwiceElutethefusionproteinbyadding10lofGlutathioneElutionBuffensptendtheSepharosebeadsandincubatefor5minatr.t.Cent

51、rifugeinamicrocentrifugefor5mintosedimenttheSepharosebeads,thentransferthesupernatanttofreshtubes.*Glutathioneelutionbuffer:10mMreducedglutathionein50mMTris-HCl(pH8.0).Dispensein1-10mlaliquotsandstoreat20Cuntilneeded.Avoidmorethanfivefreeze/thawcycles.PreparationofGlutathioneSepharose4B(forbulkmatri

52、xforbatchpurification)GentlyshakethebottleofGlutathioneSepharose4Btoresuspendthematrix.精品文檔精品文檔Useapipettoremovesufficientslurryforuseandtransfertoanappropriatecontainer/tube.(GlutathioneSepharose4Bassuppliesisapproximatelya75%slurry.Thefollowingprocedureresultsina50%slurry;Basedonthebedvolumerequir

53、ement,dispense1.33mloftheoriginalGlutathioneSepharose4Bslurrypermlofbedvolumerequired).Sedimentthematrixbycentrifugationat500xgfor5min,carefullydecantthesupernatant.WashtheGlutathioneSepharose4Bbyadding10molofcold(4C)1xPBSper1.33mloftheoriginallyslurryofGlutathioneSepharose4Bdispensed,Inverttomix.-N

54、ote:GlutathioneSepharose4Bmustbethoroughlywashedwith1xPBStoremovethe20%ethanolstoragesolution.Residualethanolmayinterferewithsubsequentprocedures.Sedimentthematrixbycentrifugationat500xgfor5min.Decantthesupernatant.Foreach1.33mloftheoriginalslurryofGlutathioneSepharose4B,and1mlof1xPBS.Thisresultsina50%slurry.Mixwellpriortosubsequentpipettingsteps.-Note:GlutathioneSepharose4Bequilibratedwith1xPBSmaybestoredat4Cforu