1Developinganefficientproteinpurificationscheme2IntroductionThreephasestrategyCombiningtechniquesPurityrequirementsCharacteristicsofthetargetproteinandcontaminantsExamplesSummaryandshortcutsDevelopinganefficientproteinpurificationscheme3ProteinPurification-AimsSufficientpurityandquantityMaintainedbiologicalactivityGoodeconomy4YieldsfromMultistepProteinPurificationsNumberofstepsYield(%)95%/step90%/step85%/step80%/step75%/step020406080100123456785InputforPurificationProtocolDevelopment

ThreephasestrategyPurificationprotocolRequiredpurityandquantityPhysical-chemicalpropertiesoftargetandmaincontaminantsSourcematerialinformationSeparationtechniqueknowledgeScoutingrunsandoptimizationEconomyandresources6ProteinPurificationAnalyticaltoolsArapidandreliableassayforthetargetproteinPuritydetermination

(e.g.SDS)Totalproteindetermination

(e.g.colorimetricmethod)7ThreePhaseStrategyPurityStepCaptureIntermediatepurificationPolishingIsolateproduct,concentrate,stabilizeRemovebulkimpuritiesAchievefinalpurity.Removetraceimpurities,structuralvariants,aggregatesetc.8Capture

ResolutionSpeedRecoveryCapacityInitialpurificationofthetargetmoleculefromcrudeorclarified sourcematerial

Concentrationandstabilization(e.g.removalofproteases)9IntermediatePurification

ResolutionSpeedRecoveryCapacity

Removalofbulkimpurities10Polishing

ResolutionSpeedRecoveryCapacity

Finalremovaloftracecontaminants,e.g.structuralvariantsof thetargetprotein

11ThreePhaseStrategy-RankingofChromatographyTechniques

Technique Capture Intermediate Polishing

GF

IEX

HIC AC

RPC

ConsiderationsLimitedsamplevolumeLimitedflowraterangeProteinligandissensitivetoharshcleaningconditionsUseoforganicsolvents,lossofbiologicalactivity12LinkingChromatographyTechniquesintoaPurificationProtocol-GeneralRulesCombinetechniqueswithcomplementaryselectivities(e.g.IEX,HICandGF).

Minimizesamplehandlingbetweenpurificationsteps(e.g.concentration,bufferexchange).

13LinkingChromatographyTechniquesTechniqueEndconditionsStartconditionsSmallsamplevolumeGFDilutedsampleBufferchange(ifrequired)LowionicstrengthIEXHighionicstrengthor

pHchangeHighionicstrengthHICLowionicstrengthSpecificbindingconditionsACSpecificelutionconditions14LinkingChromatographyTechniques

1.IEX HIC GF 2.AC GF

RPC IEX 3.HIC GF AC GF4.(NH4)2SO4 HIC IEX GF HIC GF IEX5.GF GF(desalting) AC GF15PurityRequirements Contaminantswhichdegradeorinactivatethetargetprotein(e.g. proteases),needtobereducedto“non-detectable”levels.

Contaminantswhichinterferewithsubsequentanalysesneedto bereducedto“non-detectable”levels.

Itisbetterto“over-purify”thanto“under-purify”.16Therapy

Invivostudies Crystallizationforx-raystudies N-terminalsequencing ofanunknownprotein Mostphysical-chemicalcharacterizationmethods Antigenformonoclonal antibodyproductionExtremelyhighHighModeratePurityRequirements-BriefGuidelines17TowardstheOptimalPurificationProtocol-AccountingforTargetProteinProperties(1)Targetproteinproperty

PurificationparameteraffectedStabilitywindowpHIonicstrengthCo-factorsDetergentconcentrationOrganicsolventsOther(light,oxygenetc.)IEXconditions(alsoACandRPC)HICconditionsselectionofbuffers,pH,salts,additivesbufferadditivesRPCconditionsvarious18TowardstheOptimalPurificationProtocol-AccountingforTargetProteinProperties(2)Physical-chemicalpropertiesChargeproperties(isoelectricpoint)MolecularweightPost-translationalmodificationsBiospecificaffinityTargetproteinproperty

PurificationparameteraffectedselectionofIEXconditionsselectionofGFmediumselectionofgroupspecificACmediumselectionofligandforAC19TargetProteinStabilityWindowDeterminationofasuitableammoniumsulfateconcentrationandpHscreeningrangeforHIC20TargetProteinProperties

Selectionofionexchangeconditions05678pHmoleculescharge+-Electrophoretictitrationcurveofchickenbreastmuscle

usingzymogramdetectionforcreatinekinaseTargetproteinContaminants21GProteinReceptorKinasePurificationTechniquePptHICAIEXCIEXACPurification

factorComment720240818647

Allbufferscontain

proteaseinhibitors Allpurificationsdoneat+4oC

Removalstep,main

contaminantisbound

Elutionbufferisusedas startingbufferfornextcolumn 10mghomogenousprotein

obtainedA.Tobinetal.(1996)J.Biol.Chem.271,3907-3916PorcinecerebellahomogenateRESOURCEQAmmoniumsulfate

precipitationButylSepharoseFastFlowRESOURCEsHiTrapHeparin22Reca-MannosidasePurificationfromPichiaTechniquePurification

factorComment 83mghomogenousprotein

obtainedY.-F.Liaoetal.(1996)J.Biol.Chem.271,28348-28358Capturewithstepgradient;

730mgoftotalproteinapplied63622719UFGFAIEXHICPhenylSepharoseHPQSepharoseFFSuperdex200pgUltrafiltration23DNABindingProteinPurificationTechniqueDNA-1SepharoseCIEXACACACCIEXPurification

factorComment584943 GeneralACstepforDNA

bindingproteins Removalstep,non-specific

DNAbindingactivityremovedMainpurificationstep Finalpolishing,20mgprotein

obtainedJ.Berthelsenetal.(1996)

J.Biol.Chem.271,3822-383092447 RapidcaptureHeLacellnuclear

extractsSPSepharoseHighPerformanceHeparinSepharose

FastFlowDNA-2SepharoseMonoS24

Finalpolishingandpuritycheck,

20mgobtainedMembraneProteinPurificationTechniqueACAIEXCIEXACCIEXPurification

factorComment341442

Negativestep;contaminant removed

Detergentexchange,volume

reductionbeforeAC

MainpurificationstepT.Whiteetal.(1995)

J.Biol.Chem.270,24156-241656242

Stepgradient,rapidconcentrating

capturestepPlacentaextractin

1.5%TritonX-100BlueSepharoseDEAESephacelSPSepharoseFFMuc2SepharoseMonoS25TowardsaGeneralProteinPurificationProtocolArapidmethodforobtainingmilligramquantitiesofdifferentrecombinantproteins,forinitialcharacterizationstudiesSemi-automatedin?KTAexplorer,withpre-mademethodtemplatesandBufferPrepIonexchangeSTREAMLINESPorDEAESPorQSepharoseFFHydrophobicinteractionPhenylSepharoseFF(highsub)GelfiltrationSuperdex75prepgrade26TowardsaGeneralProteinPurificationProtocol-ResultswithE.colir-ProteinsIonexchangeSTREAMLINESPorDEAESPorQSepharoseFFHydrophobicinteractionPhenylSepharoseFF(highsub)GelfiltrationSuperdex75prepgradeProtein Expression Capturestep(purifiedtohomogeneity)

AnnexinV Extracellular STREAMLINEDEAEa-Amylase Intracellular STREAMLINEDEAEanti-gp120Fab Periplasmic SPSepharoseFastFlow

27Shortcuts-RapidEstablishmentofMilligramScalePurificationProtocolsIfabiospecificligandisavailable:

useACasthemainpur