蛋白纯化方法

ProteinPurificationStrategiesCourse:MethodsinproteinchemistryRahmanM.Mahfuzur2012/01/11SLUToseperateaparticularprotinfromallotherproteinsandcellcomponentsTherearemanytypesofproteinswithinanogranismOthercomponents:nuclicacids,charbohydrates,lopids,smallmoleculesAgievenproteincouldbe0.001-20%oftotalprotein.ObjectivesProteinspurificationvariesfromonepurificationsteptomulti-stepofpurifixcationsOftenmorethanonepurificationstepisnecessarytoreachthedesiredpurity,orstepcanberepeteadifsampleisavailable.Successfulandefficientproteinpurificationdependsonappropriatemethodsselection.Methodsshouldbesequenceinalogicalmanner,whatkindsofmaterialsareavailable/handle?whathastoberemoved/completely?whatwillbetheuseoffinalproducts?whatareeconomicalconstraints?ProteinpurificationThreephasepurificationstrategies(CIPP)ThefourparametersEverytechniqueoffersabalancebetweenresolution,capacity,speedandrecoveryCaptureInitialpurificationoftargetRapidisolation,stabilization,concentrationIntermediatepurificationFurtherremovalofbulkcontaminants:otherproteins,nucleicacids,endotoxinsandviruses.Purificationandconcentration.PolishingFinalremovalofremainingtraceimpuritiesorcloselyrelatedsubstancestoachievehighpurityProteinpropertyTechniqueChargeIonexchange(IEX)Specificligandrecognition(biospecifcornonbiospecifc)Affinitychromatography(AC)SizeGelfiltration(GF)HydrophobicityHydrophobicinteraction(HIC),Reversedphase(RPC)IsoelectricpointChromatofocusingProteinpropertiesVsTechniqueIonexchangechromatography(IEX)separatesproteinswithdifferencesinsurfacecharge.basedonthereversibleinteraction,chargedproteinVsoppositelychargedcolumnIf,pHbPIPNeg.bindpositivelychargedanionexchanger;ex-MonoQcolumnWhen,pHbPIPPos.bindnegativelychargedcationexchanger;ex-MonoScolumnIEXchromatogramAffinitychromatography(AC)OnthebasisofareversibleorspecificinteractionbetweentargetandaspecifcligandBiospecific:antibodiesbindingproteins,Non-biospecific:histidinebindingproteinbindtometalIon;IMACACchromatogramGelfiltrationchromatography(GF)allowseparationofproteinswithdifferencesinmolecularsizeGFisanon-bindingmethod0.5%to2%oftotalcolumnvolume.FGchromatogramHydrophobicinteractionchromatography(HIC)separatesproteinswithdifferencesinhydrophobicitybasedonthereversibleinteractionbetweenaproteinandthehydrophobicsurfaceofachromatographymediumTechniqueVsthreephasecombinationsofchromatographicstepsIEX-HIC-GFConsideredasastandardprotocolIfnothingisknownaboutthetargetproteinuseIEX-HIC-GF.bothanionandcationexchangecouldbeusedtogetdifferentselectivitieswithinthestrategy.Thanksforlisteningme!