包涵体表达蛋白的纯化方法Purification of Expressed Proteins from Inclusion Bodies_蚂蚁淘，【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城

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包涵体表达蛋白的纯化方法Purification of Expressed Proteins from Inclusion Bodies

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JosephSambrookPeterMaccallumCancerInstituteandTheUniversityofMelbourne,AustraliaDavidW.RussellUniversityofTexasSouthwesternMedicalCenter,DallasExcerptedFromMolecularCloning:ALaboratoryManualThirdEdition

ABSTRACT
TheexpressionofforeignproteinsathighlevelsinE.colioftenresultsintheformationofinclusionbodiescomposedofinsolubleaggregatesoftheexpressedprotein.TheinclusionbodiesarerecoveredfrombacteriallysatesbycentrifugationandarewashedwithTritonX-100andEDTAtoremoveasmuchbacterialproteinaspossIBLefromtheaggregatedforeignprotein.Toobtainsolubleprotein,thewashedinclusionbodiesaredissolvedindenaturingagentsandthereleasedproteinisthenrefoldedbygradualremovalofthedenaturingreagentsbydilutionordialysis.Whereastheproceduregivenherehasbeenusedtosolubilizeinclusionbodies,eachproteinmayrequireaslightlydifferentprocedure,whichmustbedeterminedempirically.

MATERIALS
ReagentsandSolutions
CelllysisbufferI 50mMTris-Cl(pH8.0) 1mMEDTA(pH8.0) 100mMNaCl CelllysisbufferII 50mMTris-Cl(pH8.0) 10mMEDTA(pH8.0) 100mMNaCl 0.5%(v/v)TritonX-100 Deoxycholicacid,proteingrade HCl(12M)(concentratedHCl) InclusionbodysolubilizationbufferI(preparejustbeforeuse) 50mMTris-Cl(pH8.0) 1mMEDTA(pH8.0) 100mMNaCl 8Murea 0.1MPMSForPefablocSC InclusionbodysolubilizationbufferII 50mMKH₂PO₄(pH10.7) 1mMEDTA(pH8.0) 50mMNaCl KOH(10N) PMSF(phenylmethylsulfonylfluoride)(17.4mg/mlinisopropanolat-20°C)AnalternativetoPMSF,PefablocSC,isnontoxicandstableinbufferedaqueoussolutions. Tris-Cl(0.1M,pH8.5)withurea.ForuseinMethod2ofStep7.Prepare0.1MTris-Cl(pH8.5)withincreasingconcentrationsofurea(e.g.,0.5,1,2,and5M).Becauseureadecomposesinaqueoussolutions,makethesolutionfreshfromsolidureaanduseimmediately.
VectorsandHosts
E.colicells(1-literculture)expressingaproteinofinterestasaninclusionbody
EnzymesandBuffers DNaseI(1mg/mlin20mMTris-Cl[pH7.8]) Lysozyme
Gels/LoADIngBuffers 10%PolyacrylamidegelcontainingSDS 1xSDSgel-loadingbuffer 50mMTris-Cl(pH6.8) 2%(w/v)SDS,electrophoresisgrade 0.1%(w/v)bromophenolblueAdddithiothreitolfroma1Mstocktoafinalconcentrationof100mMjustbeforethebufferisusedinStep13. 2xSDSgel-loadingbuffer 100mMTris-Cl(pH6.8) 4%(w/v)SDS,electrophoresisgrade 0.2%(w/v)bromophenolblue 20%glycerolAdddithiothreitolfroma1Mstocktoafinalconcentrationof100mMjustbeforethebufferisusedinSteps7and14.
Centrifuges/Rotors/Tubes SorvallSLC-1500rotor(4°C)
AdditionalItems Glassrod(polished) pHpaper

METHOD
Centrifuge1literofthecellcultureofE.coliexpressingtheproteinofinterestat5000g(5500rpminaSorvallSLC-1500rotor)for15minutesat4°Cinpreweighedcentrifugebottles.IMPORTANT:PerformSteps2-4at4°C. RemovethesupernatantanddeterminetheweightoftheE.colipellet.Foreachgram(wetweight)ofE.coli,add3mlofcelllysisbufferI.ResUSPendthepelletbygentlevortexingorbystirringwithapolishedglassrod. ForeachgramofE.coli,add4µlof100mMPMSForPefablocSCandthen80µlof10mg/mllysozyme.Stirthesuspensionfor20minutes. Stirringcontinuously,add4mgofdeoxycholicacidpergramofE.coli. Storethesuspensionat37°Candstiritoccasionallywithaglassrod.Whenthelysatebecomesviscous,add20µlof1mg/mlDNaseIpergramofE.coli. Storethelysateatroomtemperatureuntilitisnolongerviscous(~30minutes). Purifyandwashtheinclusionbodiesusingoneofthefollowingtwomethods.Method1:RecoverinclusionbodiesusingTritonX-100 Centrifugethecelllysateatmaximumspeedfor15minutesat4°Cinamicrofuge. Decantthesupernatant.Resuspendthepelletin9volumesofcelllysisbufferIIat4°C. Storethesuspensionfor5minutesatroomtemperature. Centrifugethetubeatmaximumspeedfor15minutesat4°Cinamicrofuge. Decantthesupernatantandsetitasideforthenextstep.Resuspendthepelletin100µlofH₂O. Remove10-µlsamplesofthesupernatantandoftheresuspendedpellet.Mixeachsamplewith10µlof2xSDSgel-loadingbufferandanalyzethesamplesbySDS-polyacrylamidegelelectrophoresistodeterminewhichfractioncontainstheproteinofinterest. Ifnecessary,proceedwithStep8tosolubilizetheinclusionbodies. Method2:Recoverinclusionbodiesusingurea Centrifugethecelllysateatmaximumspeedfor15minutesat4°Cinamicrofuge.IMPORTANT:PerformStepsb,d,andfat4°C.Thefollowingstepsinvolvewashingandsolubilizationofinclusionbodieswithbufferscontainingdifferentconcentrationsofurea. Decantthesupernatant.Resuspendthepelletin1mlofH₂OpergramofE.coli.Transfer100-µlaliquotstofourmicrofugetubesandstoretheremainderofthesuspensionat4°C. Centrifugethe100-µlaliquotsatmaximumspeedfor15minutesat4°Cinamicrofuge. Discardthesupernatants.Resuspendeachpelletin100µlof0.1MTris-Cl(pH8.5)containingadifferentconcentrationofurea(e.g.,0.5,1,2,and5M). Centrifugethetubesatmaximumspeedfor15minutesat4°Cinamicrofuge. Decantthesupernatantsandsetthemasideforthenextstep.Resuspendeachpelletin100µlofH₂O. Remove10-µlsamplesofeachsupernatantandeachresuspendedpellet.Mixeachsampleandresuspendedpelletwith10µlof2xSDSgel-loadingbufferandanalyzebySDS-polyacrylamidegelelectrophoresistodeterminewhichconcentrationofureayieldsthebestrecoveryoftheinclusionbodies. Usetheappropriateconcentrationofurea,determinedinStepg,towashtheremainingpellet(fromStepb)asdescribedinthismethod. Ifnecessary,proceedwithStep8tosolubilizetheinclusionbodies. CentrifugetheappropriateresuspendedpelletsfromStep7atmaximumspeedfor15minutesat4°Cinamicrofugeandsuspendthemin100µlofinclusion-bodysolubilizationbufferIcontaining0.1mMPMSForPefablocSC(freshlyadded). Storethesolutionfor1houratroomtemperature. Addthissolutionto9volumesofinclusion-bodysolubilizationbufferIIandincubatethemixturefor30minutesatroomtemperature.CheckthatthepHismaintainedat10.7byspottingsmallaliquotsontopHpaper.Ifnecessary,readjustthepHto10.7with10NKOH. AdjustthepHofthesolutionto8.0with12MHClandstoretheadjustedsolutionforatleast30minutesatroomtemperature. Centrifugethesolutionatmaximumspeedfor15minutesatroomtemperatureinamicrofuge. Decantthesupernatantandsetitasideforthenextstep.Resuspendthepelletin100µlof1xSDSgel-loadingbuffer. Remove10-µlsamplesofthesupernatantandresuspendedpellet.Mixthesupernatantsamplewith10µlof2xSDSgel-loadingbuffer.AnalyzebothsamplesbySDS-polyacrylamidegelelectrophoresistodeterminethedegreeofsolubilization.

Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:MolecularCloning:ALaboratoryManualThirdEdition,byJosephSambrookandDavidW.Russell,©2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.15.49.

Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission.