蛋白纯化策略

1/GE/2Tag/PTMHydro-phobicityNetChargeSize/globularvolume/Ionexchangechromatography(IEX)Affinitychromatography(AC)Hydrophobicinteractionchromatographhy(HIC)Gelfiltration(GF)3ContentPlanningpurificationStrategiesExamplesSummary4PlanningpurificationDefineobjectivesCollectinformationtargetproteinstartingmaterialSelectanalyticaltechniques5pgngµgmggkg699%95-99%80%1/GE/Strategiesforproteinpurification2Tag/PTMHydro-phobicityNetchargeSize/globularvolumeIonexchangechromatography(IEX)Affinitychromatography(AC)Hydrophobicinteractionchromatographhy(HIC)Gelfiltration(GF)Specificity3ContentPlanningpurificationStrategiesExamplesSummary4PlanningpurificationDefineobjectivesCollectinformationtargetproteinstartingmaterialSelectanalyticaltechniques5pgngµgmggkgTherapeuticproteinsStructuralstudiesFunctionalstudiesMassspectroscopyDefineobjectives:ProteinamountrequiredAntigenforimmunization6Defineobjectives:PurityrequirementExtremelyhigh99%High95-99%Moderate80%TherapeuticproteinsStructuralstudiesFunctionalstudiesMassspectroscopyAntigenforimmunization2/GE/7?“over-purify”“under-purify”,,aggregatesandstructuralheterogeneity,DNA8280nm(pI)Tag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGFTag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGFTag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGF9()10,SDS-PAGE,GF,Westernblotting,MSGF,IEF:pH,//11122HNCOOH2HNCOOHHostCell2/GE/7Whatarethecontaminants?Itisbetterto“over-purify”thanto“under-purify”Ingeneral,proteaseswhichdegradeorinactivatethetargetproteininterferingcomponentsinsubsequentanalysisanduseForstructuralstudies,aggregatesandstructuralheterogeneityFortherapeuticproteins,endotoxins,genomicDNAandvirusparticles8Collectinformation:targetproteinFromaminoacidsequenceMolecularweightTheoreticalextinctioncoefficientat280nmIsoelectricpoint(pI)LiteraturesearchTag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGFTag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGFTag/PTMACHydrophobicityHICNetchargeIEXSize/globularvolumeGF9Collectinformation:startingmaterialMembranespreparationandsolubilizationofmembraneproteinsMembranesConcentration(duetolargevolume)andclarificationSecretedinmediaCentrifugation,solubilizationandrefoldingInclusionbodiesOsmoticshockorfreeze/thawmethodPeriplasmCelldisruption,clarificationCytoplasmActionsLocationincell10SelectanalyticaltechniquesSize,puritycheckSDS-PAGE,analyticalGFIdentity,proteolyticcleavageWesternblotting,MSAggregation,heterogeneityGF,IEFStability:pH,ionicstrengthsBiologicalactivityConcentrationExtinctioncoefficient,ColorimetricproteinassayActivity/functionEnzymeassay,varioustechniquesCharacteristics/propertiesTechniques11PurificationstrategiesWorkflowinproteinpurificationSamplepreparationThreestagestrategyLinkingchromatographictechniques12ExpressionSamplepreparationChromatographicpurificationStorageEndapplicationNaturalsourceRecombinantsource2HNCOOH2HNCOOHHostCellWorkflowinproteinpurification3/GE/13,,E.coli()10000g10-15min14:CiPP1.Capture2.Intermediate3.Polishing050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120ml151.2.3.050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120ml161.2.3.050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120ml1710µmMonoQ™5/50GLRESOURCEQ™1ml15µmSOURCE™QXK16x50mm30µmQSepharose™FF16x50mm90µm34µmQSepharose™HP16x50mm183/GE/13SamplepreparationAddnucleasesPrecipitationbystreptomycinsulfate,polyethyleneimine,protaminesulfateHighviscosityMayblockresinsNucleicacids(NA)AddinhibitorsRemovebyACRapidlyperformfirstpurificationstepWorkatlowtemperaturesSelectproteasedeficientE.colistrainsDegradestargetmoleculesProteases(Phosphatases)Centrifuge10-15min10000gMayuseorganicsolventextractionMayblockresinsMaycauseaggregationLipidsFilterorcentrifugeClogscolumnParticulatematterPreventiveactionHarmfuleffectContaminant14Threestagestrategy:CiPPPurityNumberofsteps1.Capture2.Intermediate3.Polishing050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120mlSamplepreparation15ThreestagestrategyPurityNumberofstepsRemovetraceimpuritiesIsolateproductConcentrateStabilizeRemovebulkimpurities1.Capture2.Intermediate3.Polishing050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120mlSamplepreparation16ThreestagestrategyPurityNumberofstepsRemovetraceimpuritiesIsolateproductConcentrateStabilizeRemovebulkimpurities1.Capture2.Intermediate3.Polishing050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120mlSpeedCapacityResolutionCapacityResolutionRecoverySamplepreparation17ParticlesizeandresolutionPuritySamplepreparation10µmMonoQ™5/50GLRESOURCEQ™1ml15µmSOURCE™QXK16x50mm30µmQSepharose™FF16x50mm90µm34µmQSepharose™HP16x50mmNumberofsteps18ThreestagestrategyResolutionSpeedRecoveryCapacity4/GE/19IEXHICGFHICGFIEX(NH4)2SO4GFGFACIEXACAC20pHIEXACHIC050100150mAu020406080100120ml050100150mAu020406080100120ml050100150mAu020406080100120mlGF21100806040200%5101595%/90%/85%/70%