AliquotofCellPelletafterInduction

Theideaistoaliquotcellsafterinduction,andkeepat-80ºCenoughcellpelletsamplesforoptimizationofsmallscalepurificationprocedureandfurtherscale-up.Onceyousetupthebestpurificationconditionsatlowscale,youcanscale-uptheprocedure.

EquilibrationofGlutathioneAgarose

Place20ulbeads(40ulsuspension)ofglutathioneagarosein1.5mlplastictube.

Washwith2x1.5mlH₂Oand2x1.5mllysisbuffer(washing:mix,spin3min3500rpm,dischargesupernatant).

ProteinExtraction

Resuspendpelletof10mlcellculturein1mllysisbuffer(or100mlbacterialcultureforverylowexpressionlevel).

SuggestedLysisbuffer:140mMNaCl;2.7mMKCL;10mMNa₂HPO₄;1.8mMKH₂PO_4;pH7.3(PBS)optional0.02%NaN₃(azide)optionalproteaseinhibitors

Optionaladditivestothelysisbuffera)1mMPMSForproteaseinhibitorcocktail1:200(cocktailforbacterialcells#P-8849fromSigma)b)Dnase100U/mlor25-50ug/ml(SIGMADN-25).Incubate10min4°Cinthepresenceof10mMMgCl₂.c)Lysozime0.2mg/ml.Incubate10min4°C.d)ßME,DTTorDTEupto10mMforproteinswithmanycysteines.e)0.1-2%TritonX-100,NP40;oranyotherdetergentthatdonotaffecttheBIOLOGicalactivityofyourprotein.

Sonicateinicebucket3x10secormoreifthecellsarenotcompletelydisrupted(Lysisiscompletewhenthecloudycellsuspensionbecomestranslucent.Avoidproteindenaturationbyfrothing).

Spin5min13000rpm4°C.Separatesolubleproteins(supernatant)frominsolubleorinclusionbodiesproteins(pellet).Usesupernatantfornextstep.Keepsampleof40ulofsupernatantforPAGE-SDS:solubleproteins

Resuspendpelletinanother1mllysisbufferandkeepsampleof40ulforPAGE-SDS:insolubleproteins,orunlysedcells.

ProteinPurification

1)Mixsupernatantoflaststepgentlywiththeequilibratedresin60minat4°C.

2)Spin3min3500rpm4°C.Dischargesupernatantandkeepsampleof40ulforPAGE-SDS:unboundproteins(thismaterialcouldbeuseagainincaseofoverloADIng).

3)Washbeadswith2x1.5mlPBS(washing:mix,spin3min3500rpm,keepsupernatantaside).Keepsampleof40ulforPAGE-SDSofeachwashing.

4)Eluterecombinantproteinwith3x50ulelutionbuffer:50mMTrisHClpH8.010mMreduceglutathione(mixgently,incubateatRTfor5minforeachelution,spin3min3500rpm,keepsupernatantaside).Keepsampleof40ulforPAGE-SDSofeachelution.

5)Resuspendbeadsin50ulH₂O+20ul5xsamplebuffer.Mixandspin.Keepsampleof40ulforPAGE-SDS:proteinnoteluted(orSDSextractedbeads).

6)RunonPAGE-SDS:crudesupernatant;resuspendedpellet;unbound,washings,elutions,andSDSextractedbeads.MWofGSTalone:29000

Analysisofresults-Troubleshooting

Expectover-expressedproteintobefoundonlyinthecrudesupernatantandintheelutionoftheGlutathioneagarose.

Ifmostoftheproteinremainsinsolubleafterextraction,trya)TochangelysisbufferbyaddingßME,DTT,glycerol,detergentsormoreNaCl.Ifonlypartofitisinsoluble,b)Orre-extractpelletwithmorebuffer,c)Orusemorelysisbufferduringextraction,d)Orperformamoreintensivesonication,e)Orincubatewithlysozymebeforesonication.f)Ortrythedenaturatingprotocolextraction(4-6Mguanidine-HCl;4-8Murea;alkalinepH>9.0;0.5-2%TritonX-100;0.5-2%N-lauroylsarcosineorotherdetergents)

IfproteindoesnotbindtotheGlutathioneagarose,thereareseveraloptionstochoose:a)ChecktheGlutathioneagarose:bindingofacellsonicatecontainingonlyGSTb)Ifonlypartiallybound,usemoreresin,orbindforlongertime(Thelongerthedurationofpurification,thegreatertheriskofproteindegradation).c)Add1-10mMDTTpriortocelllysis(sometimescanincreasesignificantlythebinding);ortryadditivesasglycerol,detergentsormoreNaCld)Purifyproteinunderdenaturatingconditions.e)Movetagtotheoppositeendoftheprotein.

Iffusionproteinispoorlyeluted,try:a)Decreaseflowrate,ortryovernightelution(Thelongerthedurationofpurification,thegreatertheriskofproteindegradation)b)Increaseconcentrationofglutathione.First15mMandthen20-40mM.c)Increaseionicstrengthto0.1-0.2MNaCl.d)Increasethevolumeofelutionbuffere)Addanon-ionicdetergent(as0.1%TritonX-100or2%N-octylglucoside)toreducenon-specifichydrophobicinteractionsthatmaypreventsolubilizationorelutionofthefusionprotein.

Ifmultipleproteinsbandsareseenintheelutiontry:a)Ifyoususpectproteindegradation(youcancheckpreviouslywithwesternblotusingantibodiesagainstGSTorthefusionprotein)trytoworkallthetimeat4°Canduseproteaseinhibitorsduringlysis.(Thelongerthedurationofpurification,thegreatertheriskofproteindegradation)b)Decreaseresinvolume(allowshighercompetitionbetweenfusionproteinandcontaminantsforthesamesitesontheresin)c)Increaseionicstrengthto0.1-2MNaClorusedetergentsthatnotdestroytheproteinactivityduringwashingstepd)Increasethevolumeofthewashingstep.e)Consideranadditionalpurificationstepbeforeorafterpurification.