GST融合蛋白纯化方法 Purification of GST Fused Proteins
来自 : 蚂蚁淘
Abstract:ManypeoplehaveventedoutfrustrationoverinsolubleGST-fusedproteins.ThisisaprotocolforenzymaticallyactivesolubleGST-fusedproteins.AllGST-fusedproteinsarerenderedsolublewiththistechniquethoughenzymeactivitiycanrangefrom30-90%.
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MaterialsandReagents
STEBuffer10mMTris-HCl,pH8.01mMEDTA150mMNaCl
Lysozymesolution10mg/mlinwater(makefresh)
PBS
ElutionBuffer50mMTris.Cl,pH9.020mMGSH
10%SarkosylinSTEBuffer
10%TritonX-100inSTEBuffer
1MDTT
100mMIPTG
Procedure
Day1
Setupanovernightculturein50ml2XTYwith150mg/mlofampicillin.
Day2
Seed5mlofovernightcultureto500ml2XTYwith150mg/mlofampicillin.
Growat37oCtoanA600of0.6to0.8.
Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.LowerIPTGconcentrationsandlowergrowingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.
Pelletcellsbycentrifugingat3000g,4oCfor10min.DecantmediaandresUSPendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4oCfor10min.DecantPBS. Thisisaconvenientpointtostopandtostorepelletsat-80oC.Elsecontinuetolysecells.
Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.
Resuspendpelletin10mloficecoldSTEBuffer.
Add100mloffreshlypreparedlyozymesolution,incubateonicefor15min.Justbeforesonication,add100mlof1MDTTand1.4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.
Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthepellet.Add4mlof10%TritonX-100andtopupwithSTEBufferto20ml.TheeffectiveconcentrationofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.
Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1hrwithagitation.Topreparethe50%slurry,shakeupthemediaandPipette2mltoa50mltube.Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.
Washthebeadswith3X50mlofPBS.Finallyresuspendin5mlofPBS.Pourtoadispo-column.Washthe50-mlconicaltubewithanadditional5mlofPBS.Poolwiththefirst5mlinthedispo-column.Towash,usethesamecentrifugationtechniqueforpreparingthebeads.Whentransferringbeadstocolumn,donotpipettebutpour.Thebeadstendtosticktopipettetips.
Ifdesired,elutewith10x1mlfractionsofElutionBuffer.DeterminedesiredfractionswithSDSPAGE.
Reference
FrangioniandNeel.Anal.Biochem.210,179-187(1993)
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