蛋白纯化方法.ppt

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1、ProteinPurificationStrategiesCourse:MethodsinproteinchemistryRahmanM.Mahfuzur2012/01/11SLUToseperateaparticularprotinfromallotherproteinsandcellcomponentsTherearemanytypesofproteinswithinanogranismOthercomponents:nuclicacids,charbohydrates,lopids,smallmoleculesAg

2、ievenproteincouldbe0.001-20%oftotalprotein.ObjectivesProteinspurificationvariesfromonepurificationsteptomulti-stepofpurifixcationsOftenmorethanonepurificationstepisnecessarytoreachthedesiredpurity,orstepcanberepeteadifsampleisavailable.Successfulandefficientprote

3、inpurificationdependsonappropriatemethodsselection.Methodsshouldbesequenceinalogicalmanner,whatkindsofmaterialsareavailable/handle?whathastoberemoved/completely?whatwillbetheuseoffinalproducts?whatareeconomicalconstraints?ProteinpurificationThreephasepurification

4、strategies(CIPP)ThefourparametersEverytechniqueoffersabalancebetweenresolution,capacity,speedandrecoveryCaptureInitialpurificationoftargetRapidisolation,stabilization,concentrationIntermediatepurificationFurtherremovalofbulkcontaminants:otherproteins,nucleicacids

5、,endotoxinsandviruses.Purificationandconcentration.PolishingFinalremovalofremainingtraceimpuritiesorcloselyrelatedsubstancestoachievehighpurityProteinpropertyTechniqueChargeIonexchange(IEX)Specificligandrecognition(biospecifcornonbiospecifc)Affinitychromatography

6、(AC)SizeGelfiltration(GF)HydrophobicityHydrophobicinteraction(HIC),Reversedphase(RPC)IsoelectricpointChromatofocusingProteinpropertiesVsTechniqueIonexchangechromatography(IEX)separatesproteinswithdifferencesinsurfacecharge.basedonthereversibleinteraction,chargedp

7、roteinVsoppositelychargedcolumnIf,pHb>PIPNeg.bindpositivelychargedanionexchanger;ex-MonoQcolumnWhen,pHb

8、tandaspecifcligandBiospecific:antibodiesbindingproteins,Non-biospecific:histidinebindingproteinbindtometalIon;IMACACchromatogramGelfiltrationchromatography(GF)