Gel-Filtration Chromatography - SpringerLink

Gel-filtration chromatography is a popular and versatile technique that permits the effective separation of proteins and other biological ... Skiptomaincontent Advertisement SearchSpringerLink Search DownloadbookPDF DownloadbookEPUB Gel-FiltrationChromatography DownloadbookPDF DownloadbookEPUB AbstractGel-filtrationchromatographyisapopularandversatiletechniquethatpermitstheeffectiveseparationofproteinsandotherbiologicalmoleculesinhighyield.Here,thebasisofthemethodisdescribedandtypicalmatrixtypesarecontrasted.Theselectionofsuitableoperatingconditionsandapplicationsofthemethodarealsodiscussed.KeywordsGel-filtrationchromatographyGel-permeationGel-exclusionSize-exclusionMolecular-sieveOperatingconditionsSeparationsMolecularmassestimationSize-exclusion­reactionchromatography DownloadprotocolPDF SpringerNatureisdevelopinganewtooltofindandevaluateProtocols. Learnmore 1IntroductionGel-filtrationchromatographyisaformofpartitionchromatographyusedtoseparatemoleculesofdifferentmolecularsizes.Thistechniquehasalsofrequentlybeenreferredtobyvariousothernames,includinggel-permeation,gel-exclusion,size-exclusion,andmolecular-sievechromatography.Thebasicprincipleofgel-filtrationisrelativelysimple.Moleculesarepartitionedbetweenamobilephaseandastationaryphasecomprisingaporousmatrix(ofdefinedporosity)asafunctionoftheirrelativesizes.Acolumnconstructedofsuchamatrix,typicallyinbeadform,willhavetwomeasurableliquidvolumes:theexternalvolume,consistingoftheliquidbetweenthebeads,andtheinternalvolume,consistingoftheliquidwithinthebeads.Theexternalvolumeisusuallyreferredtoasthevoidvolume(V 0),whilethesumoftheexternalandinternalvolumesisthetotalvolume(V t).Followingsampleapplication,moleculeslargerthantheporesofthestationaryphasematrixwillbeexcludedfromtheinternalvolumewithinthebeads.Theywill,therefore,migratequiterapidlythroughthecolumn,emergingatV 0,whilemoleculessmallerthanthematrixpores(aswellasthoseintermediateinsize)willequilibratewithboththeexternalandinternalliquidvolumes,causingthemtomigratemuchmoreslowlyandemergeatavolume(V e)greaterthanV 0.Moleculesare,therefore,elutedinorderofdecreasingmolecularsize.Theelutionvolume,V e,ofaparticularmoleculedependsonthefractionofthestationaryphaseavailabletoitfordiffusion.ThiscanberepresentedbytheconstantK dorK av(alsoreferredtoasthepartitioncoefficient).Therefore: $${V_{\text{e}}}={V_0}+{K_{\text{av}}}\left({{V_{\text{t}}}-{V_0}}\right)$$ Rearrangingthisequationgives: $${K_{\text{av}}}=\frac{{\left({{V_{\text{e}}}-{V_0}}\right)}}{{\left({{V_{\text{t}}}-{V_0}}\right)}}$$ Inadditiontomolecularsizeormass,theflowbehaviourofmoleculesthroughagel-filtrationcolumnisalsoafunctionoftheirmolecularshape,or,tobemoreprecise,hydrodynamicdiameter.Thisisdefinedasthediameterofthesphericalvolume(hydrodynamicvolume)createdbyamoleculeasitrapidlytumblesinsolution.Whenperforminggel-filtrationchromatography,onegenerallyassumesthatallthemoleculeswithinamixturehavethesamesymmetricalshape,sothattheorderofelutionwillbeoneofdecreasingmolecularweight.Althoughthisisanacceptableassumptioninmostcases,onemustbearinmindthattheoperativemoleculedimensionduringgel-filtrationisthehydrodynamicvolumeand,assuch,anasymmetricalmoleculewillappeartoelutewithanabnormallyhighmolecularweightcomparedwithasymmetricalmoleculeofsimilarmolecularweight.Whenseparatingoutproteins,forexample,theusualassumptionisthatalltheproteinsinthemixtureareglobularproteins.Asymmetricalproteins(fibrousproteinsandcertainglycoproteins),however,willappeartoelutewithanabnormallyhighmolecularweightcomparedwithglobularproteinsofsimilarmolecularweight.1.1SelectionofOperatingConditionsVariousfactorsshouldbeconsideredwhendesigningagel-filtrationsystem.Theseinclude:(1)matrixchoice;(2)samplesizeandconcentration;(3)columnparameters;(4)choiceofeluent;(5)effectofflowrate;and(6)columncleaningandstorage.1.1.1MatrixChoiceCommonlyusedgel-filtrationmatricesconsistofporousbeadscomposedofcross-linkedpolyacrylamide,agarose,dextran(seeTable1)orcombinationsofthese,andaresuppliedeitherinsuspendedformorasdriedpowders.Thematrixshouldbecompatiblewiththepropertiesofthemoleculesbeingseparatedanditsstabilitytoorganicsolvents,pH,andtemperatureisalsoanimportantconsideration.Underseparationconditions,matricesshouldbeinertwithrespecttothemoleculesbeingseparatedinordertoavoidpartialadsorptionofthemoleculestothematrix,notonlyretardingtheirmigrationthroughthecolumn,butalsoresultingin“tailed”peaks(e.g.see(1)).Table1Somemediaforgel-filtrationFullsizetable Whenchoosingasuitablematrix,onewithamolecularmassfractionationrange,whichwillallowthemoleculeofinteresttoeluteafterV 0andbeforeV t,shouldbeselected.Themostsuitablefractionationrange,however,willbedictatednotonlybythemolecularmassofthetargetmolecule,butalsobythecompositionofthesamplebeingappliedtothecolumn.Therefore,thebestseparationofmoleculeswithinasamplehavingsimilarmolecularmassesisachievedusingamatrixwithanarrow­fractionationrange.1.1.2SampleSizeandConcentrationMaximumresolutioningel-filtrationchromatographydependsontheapplicationofsampleinasmallvolume,typically1–5%ofthetotalbedvolume.Forthisreason,gel-filtration­chromatographyhasaninherentlowsample-handlingcapacityand,accordingly,shouldbeperformedquitelateinapurificationprocedurewhenthenumbersofdifferentmoleculesinasamplearerelativelylow.Theconcentrationofasamplewhichcanbeappliedtothecolumnwillbelimitedbytheviscosityofthesample(whichincreaseswiththesampleconcentration)relativetothatoftheeluent.Ahighviscositywillresultinirregularsamplemigrationthroughthecolumnwithsubsequentlossofresolutionand,insomeinstances,willreducethecolumnflowrate.Whenseparatingproteinsbygel-filtration,thesampleshouldnothaveaproteinconcentrationinexcessof20mg/mL.1.1.3ColumnParametersMaximumresolutioningel-filtrationchromatographyisobtainedwithlongcolumns.Theratioofcolumndiametertolengthcanrangefrom1:20upto1:100.1.1.4ChoiceofEluentAsgel-filtrationchromatographyseparatesmoleculesonlyonthebasisoftheirrelativesizes,thetechniqueiseffectivelyindependentofthetypeofeluentused.Elutionconditions(pH,essentialions,cofactors,proteaseinhibitors,etc.)whichwillcomplementtherequirementsofthemoleculeofinterestshould,therefore,beselected.However,theionicstrengthoftheeluentshouldbehighenoughtominimiseprotein–matrixandprotein–proteinassociationsbyelectrostaticorvanderWaalsinteractions(e.g.see(1)).Theadditionof0.1MNaClorKCltotheeluenttoavoidtheseinteractionsisquitecommon.1.1.5EffectofFlowRateLowflowratesoffermaximumresolutionduringgel-filtrationchromatography,sinceflowrateandresolutionareinverselyrelated.Theoptimumflowrateforresolutionofproteinsisapproximately2mL/cm2/h,althoughmuchhigherflowratescanbeused,particularlywithrigidmatricessuchastheSephacrylHRrangefromGEHealthcare(30mL/cm2/h).Unfortunately,lowflowratesmeanlongerseparationtimes.Therefore,acompromisebetweendesiredresolutionandspeedmustbedecidedupon.1.1.6ColumnCleaningandStorageMostgel-filtrationmatricescanbecleanedwith0.2Msodiumhydroxideornon-ionicdetergents.Whenleftunusedforlongperiodsoftime,matricesshouldbestoredat4°Cinthedarkinthepresenceofanantimicrobialagent(0.02–0.05%[w/v]sodiumazideor20%[v/v]ethanol).2ApplicationsofGel-FiltrationChromatographyOneoftheprincipaladvantagesofgel-filtrationchromatographyisthatseparationcanbeperformedunderconditionsspecificallydesignedtomaintainthestabilityandactivityofthemoleculeofinterestwithoutcompromisingresolution.Absenceofamolecule–matrixbindingstepalsopreventsunnecessarydamagetofragilemolecules,ensuringthatgel-filtrationseparationsgenerallygivehighrecoveriesofactivity.Thisseparationtechnique,however,isnotwithoutitsdisadvantages.Whenseparatingproteinsbygel-filtrationchromatography,proteolysis,forexample,becomesanincreasingproblem:thetargetproteinfrequentlybecomesanabundantsubstrateforproteasesthatmayalsobepresentinthemixture,leadingtoreducedrecoveryofactivity.Becauseofthelargesizeof­gel-filtrationcolumns,largevolumesofeluentareusuallyrequiredfortheiroperation,oftencreatingexcessiverunningcosts.Gel-filtrationalsohasaninherentlowresolutioncomparedwithotherchromatographictechniques,becausenoneofthemoleculesareretainedbythecolumnandnon-idealflowoccursaroundthebeads.Inaddition,thistechniquehasalowsample-handlingcapacitydictatedbytheneedtooptimiseresolution.Despitethesedisadvantages,gel-filtrationchromatographystilloccupiesakeypositioninthefieldofbiomoleculeseparationbecauseofitssimplicity,reliability,versatility,andeaseofscale-up.2.1SeparationofProteinsandPeptidesBecauseofitsuniquemodeofseparation,gel-filtrationchromatographyhasbeenusedsuccessfullyinthepurificationofliterallythousandsofproteinsandpeptidesfromvarioussources.Theserangefromtherapeuticproteinsandpeptides,whichtogetherconstituteamultibillioneuroworldwidemarket,toenzymesandproteinsforthebrewing,food-processing,anddiagnosticsindustries;someexamplesofeachtypearecitedbelow.Recombinanthumangranulocytecolonystimulatingfactor(rhG-CSF)wasrefoldedfrominclusionbodiesinhighyield,withgreatsuppressionofaggregatesformation,byurea-gradientsize-exclusionchromatographyonaSuperdex75column(2).Asimilartechniquewasusedtopurifyhumaninterferon-γ,solubilisedfrominclusionbodiesby8Murea,toaspecificactivityof12,000,000IU/mgwithproteinrecoveryof67%(3).Luteinisinghormone(LH)waspurified46-foldfromacrudepituitaryextractbygel-filtrationontwoSephacrylS-200columns.ThemethodexploiteddifferentialbindingofLH(inthecrudeextract)tobluedextranforthefirstchromatographystep.Beforethesecondstep,additionofhighsaltreleasedLHfromthebluedextran,enablingeffectivepurification(4).Fusionferritin(heavy-chainferritinpluslight-chainferritin)hasalsobeenpurifiedbyurea-gradientgel-filtration.Inthiscase,fusionferritinsolubilisedfrominclusionbodieswith4Mureawasappliedtothecolumn.Refoldingenhancerswereincludedintheurea-diluentbufferthatwassubsequentlyappliedtothecolumntoproduceproperlyfoldedfusionferritinmultimers(5).Acontinuousrotatingannularsize-exclusionchromatographysystempermittedthepurificationofcrudeporcinelipasewithproductivityofapproximately3mglipasepermggelperhourandanactivityrecoveryofalmost99%(6).Amongfood-useproteins,henegglysozymehasbeensuccessfullyrefoldedusingbothacrylamide-anddextran-basedgelcolumns(SephacrylS-100andSuperdex75,respectively)(7).Gel-filtrationhasalsoprovenusefulforthepurificationofthewheyproteinsalpha-lactalbuminandbeta-lactoglobulinfromaqueoustwo-phasesystems(8).2.2SeparationofOtherBiomoleculesCarbohydratesrepresentaplentiful,butsofaronlyscarcelyexploited,reservoirofunique,multifunctionalbiopolymerswhichcanbereadilyfractionatedbygel-filtrationchromatographyonthebasisoftheirrelativesizes(e.g.see(9,10)).Variousproblems,however,havelimitedthedevelopmentofgel-filtrationmethodsforoligosaccharides.First,manyofthecommerciallyavailablegel-filtrationmatricesarethemselvescarbohydrates(e.g.Sephadex,Sepharose,etc.,manufacturedbyGEHealthcare),sheddingmilligramquantitiesofheterodispersecarbohydratepolymersintothemobilephase.Second,non-specificinteractionswithmatrixmaterialsarecommon,sincesugarsareessentiallyamphipathicwithahydrophobicringstructureandhydrophilicfunctionalgroups.Despitetheseproblems,however,gel-filtrationchromatographystillremainsanimportantoptionforthepurificationofcomplexoligosaccharides.Gel-filtrationchromatographyhasformanyyearsbeenusedtoseparatevariousnucleicacidspeciessuchasDNA,RNA,andtRNAaswellastheirconstituentbases,adenine,guanine,thymine,cytosine,anduracil.LinearphagelambdaDNAandcirculardouble-strandedphageM13DNA,forexample,canbecompletelyseparatedfromchromosomalDNAandRNAbygel-filtrationonSephacrylS-1000Superfine(11).PlasmidDNAcanalsobepurifiedbygel-filtration(12),althoughmoderncommercialkitsoftenuseacentrifugalspincolumnformatforgreaterconvenience.Onerecentstudydescribesthenoveluseoftwogel-filtrationsteps,onebeforeandoneafterareverse-phaseoperation,topurifyplasmidDNAfromaclarifiedalkalineEscherichiacolicelllysate(13).2.3SeparationofCellsandVirusParticlesCellsofdifferentsizescanbeefficientlyseparatedfromoneanotherusinggel-filtrationchromatography.Methodshavebeendeveloped,forexample,toseparatebotherythrocytes(14)andplatelets(15)fromblood(mostworkersnowpreferdensity-gradientmediasuchasPercoll™orFicoll™,trademarksofGEHealthcare,fortasksofthisnature).Size-exclusionchromatographywasuseddownstreamofexpandedbedadsorptionchromatographytorecoveractiverecombinanthepatitisBcoreantigen(HbcAg)in45%yieldwithapurificationfactorof4.5(16).ASephacrylS-1000SFprovedtobeeffectiveandeconomicalinthepurificationofrecombinantBombyxmorinucleopolyhedrosisvirusdisplayinghumanpro-reninreceptor(17).SephacrylS-1000gel-filtrationchromatographygavemoreeffectivepurificationofturkeycoronavirusfrominfectedturkeyembryosthandiduseofasucrosegradient(18).2.4GroupSeparationsByselectingamatrixpore-sizewhichcompletelyexcludesallthelargermoleculesinasamplefromtheinternalbeadvolume,butwhichallowsverysmallmoleculestoenterthisvolumeeasily,onecaneffectagroupseparationinasingle,rapidgel-filtrationstepwhichwouldtraditionallyrequiredialysisforupto24htoachieve.Groupseparationcanbeused,forexample,toeffectbufferexchangeswithinsamples,fordesaltingoflabilesamplespriortoconcentrationandlyophilisation,toremovephenolfromnucleicacidpreparations,andtoremoveinhibitorsfromenzymes(e.g.see(19)).2.5MolecularMassEstimationGel-filtrationchromatographyisanexcellentalternativetoSDS-PAGEforthedeterminationofrelativemolecularmassesofproteins,sincetheelutionvolumeofaglobularproteinislinearlyrelatedtothelogarithmofitsmolecularweight(20).Onecanprepareacalibrationcurveforagivencolumnbyindividuallyapplyingandelutingatleastfivesuitablestandardproteins(inthecorrectfractionationrangeforthematrix)overthecolumn,determiningtheelutionvolumeforeachproteinstandard,andplottingthelogarithmofmolecularweightversusV e/V 0.Whenaproteinofunknownmolecularweightisappliedtothesamecolumnandelutedunderthesameconditions,onecanusetheelutionvolumeoftheproteintodetermineitsmolecularweightfromthecalibrationcurve.2.6Size-ExclusionReactionChromatography:ProteinPEGylationCovalentattachmentofPEG(polyethyleneglycol;“PEGylation”)toaproteincanattenuateitsantigenicityand/orextenditsbiologicalhalf-lifeorshelflife.Size-exclusionreactionchromatography(SERC)permitsonetocontroltheextentofareaction(suchasPEGylation)thataltersmolecularsizeandtoseparatereactantsandproducts.InSERC,injectionofreactantsontoasize-exclusionchromatographycolumnformsamovingreactionzone.Reactantsandproductspartitiondifferentlywithinthemobilephase,leadingtodifferentflowratesthroughthecolumn.Thus,productsareremovedselectivelyfromthereactionzone,shorteningtheirresidencetimeinthereactionzoneandseparatingthemintothedownstreamsectionofthecolumn.InPEGylation,additionofPEGgroupstotheproteinsignificantlyincreasesmolecularsize,allowingtheuseofSERCtoobtainadominantfinalPEGylatedproteinsizeinhighyield.Theprinciplewassuccessfullydemonstratedusingtwomodelproteins,alpha-lactalbuminandbeta-lactoglobulin(21).3ConclusionDespiteitsdisadvantagesofsampledilutionandtheneedforalowratioofsamplevolumetocolumnvolume,gel-filtrationremainsapopularseparationmethodduetoitsversatility,thewiderangeofmatricescommerciallyavailable,andthemildconditionsofoperation.Itishopedthatthisarticlehasgiventhereadersomegraspofthetechnique’swiderangeofapplicationsandhowtochooseappropriateconditionsforitsgainfuluse.Ausefulhandbookongel-filtrationisavailablefromGEHealthcare’sLifeSciencesdivisionthroughthefollowingURL:http://www6.gelifesciences.com/aptrix/upp00919.nsf/content/LD_153206006-R350.OtherusefulchromatographyhandbooksmaybeaccessedthroughlinksfromURL:http://www6.gelifesciences.com/aptrix/upp01077.nsf/Content/service_and_support∼documents_and_downloads∼handbooks. 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CiaránÓ’Fágáin.EditorinformationEditorsandAffiliationsNationalCentreforSensorResearch,SchoolofBiotechnology,DublinCityUniversity,Dublin,9,IrelandDermotWallsNationalCentreforSensorResearch,SchoolofBiotechnology,DublinCityUniversity,Dublin,9,IrelandSinéadT.LoughranRightsandpermissionsReprintsandPermissionsCopyrightinformation©2011SpringerScience+BusinessMedia,LLCAboutthisprotocolCitethisprotocolÓ’Fágáin,C.,Cummins,P.M.,O’Connor,B.F.(2011).Gel-FiltrationChromatography. In:Walls,D.,Loughran,S.(eds)ProteinChromatography.MethodsinMolecularBiology,vol681.HumanaPress.https://doi.org/10.1007/978-1-60761-913-0_2Downloadcitation.RIS.ENW.BIBDOI:https://doi.org/10.1007/978-1-60761-913-0_2Published:08October2010 PublisherName:HumanaPress PrintISBN:978-1-60761-912-3 OnlineISBN:978-1-60761-913-0eBookPackages:SpringerProtocols DownloadbookPDF DownloadbookEPUB