Protein A, Protein G, and Protein 原理及应用

Chapter24AnalysisandPurificationofAntibodyFragmentsUsingProteinA,ProteinG,andProteinLRemkoGriepandJohnMcDougall24.1IntroductionToday,monoclonalantibodies(mAbs)formthelargestcategoryofbiopharmaceu-ticalsinclinicaltrials,andtheirnumberisexpandingrapidly(DataMonitor2007a,b).Theantibodiesorfunctionalantibodyfragmentsarebeingproducednotonlyinartificialproductionsystemssuchasmammaliancells,yeast,bacteria,andplantcellsbutalsointransgenicanimalssuchasgoats,sheep,lessoftheproductionmethod,llproteins,cellculturemediaadditives,DNA,andendotoxinshavetoberemovedfromthemer,antibodyaggregates,clippedandlowmolecularweightspecies,lproteinswithaninherentaffinityforimmunoglobulins(Ig)oleculesincludeprotein-A,derivedfrom¨quist1966);protein-G,derivedfromaStaphylococcusaureus(ForsgrenandSjo¨rkandKronvall1984);andfinallyprotein-L,derivedgroup-CStreptococcus(Bjo˚kerstro¨mandBjo¨rk1989;Housdenetal.2003,fromPeptostreptococcusmagnus(A2004).Theyallcontainrepetitive55–76aminoacidresidues(Fig.24.1)thatmediatetheactualIgbinding(Kasternetal.1992).Therecombinantprotein-Lcanbeproducedatayieldofupto3g/dsahighlypure,stable,andactiveprotein-Lfractionafterpurification,whichisbindingefficientlytomostofthehumanantibodiesoftheKappaisotype(Fig.24.2).Protein-Gbindsnoore,ademicgroupshavealsoreportedtheuseofgeneticallyfusedprotein-LG(Kihlbergetal.1996;Harrisonetal.2008)orprotein-AG(Eliassonetal.1988;(*)all´en21,Oslo3490,NorwayAffitechAS,Gaustadallee-mail:@affi¨bel(eds.),AntibodyEngineeringVol.2,10.1007/978-3-642-01147-4_24,#Springer-VerlagBerlinHeidelberg2010301

bers,indicat-ingtheaminoacidsofthebeginningofeachdomain,edarethesignalpeptide(SP),thesignalpeptidecleavagesiteisindicatedbythearrow,theNH2-terminal(A),therepeatedunitswithIg-bindingactivity(B1–B5),thespacerregion(S),therepeats(C),thewallspanningdomain(W),andthetransmembraneregion(M).Therecombinantprotein-LconsistsoffourIg-bindingdomains(B1–B4),whichcanbindtotheKapparegionwithoutinterferingwiththeantigen-bindingsiteoftheimmunoglobulinBergmann-Leitneretal.2008)andprotein-LA(Svenssonetal.1998)formonoclo-nalantibodypurifideedobtainedbroaderfunctionalligandsbecaulityofprotein-A,-G,or-Ltomaintaintheirfunctionality,onconjugationwithfluoro-chromes,enzymes(Fig.24.3a),orgoldparticles,makesthemhighlyvaluablesecondaryreagentsforthedetectionofprimaryantibodiesinELISA,immunohis-tochemistry,flowcytometry,n-AmainlybindstotheFc-regionoftheIgGfromseveralhumanisotypes(Table24.1)butonlytoasinglevariableregionoftheheavy-chainfamily(Starovasniketal.1999).Incontrast,protein-LbindstomostofthehumanKappalight-chainsofthekI,kIII,omprise55–60%ofallIgA,IgE,andIgMantibodiesinthehumanserum(Solomon1976)andcanthusbeusedtopurifyallmonoclonalantibodiesofthoseKappasub-types(Nilsonetal.1992),withouttheneedtogeneticallyengineeraffinity-tagsontotheproteinofinterest(Devauxetal.2001;Dasetal.2005;Cossinsetal.2007).ThekantibodiesdescribedinFig24.3bwereoriginallyderivedfromalargehumanunbiasedantibodyphagelibrary(Løsetetal.2005)andsixoutofthetenkantibodiesstronglyreactwithprotein-L(Fig.24.3b).Theseauthorsalsodemon-stratedthatpreselectiondsphage-antibodieswithimprovedfunctionality,aseachphageisactuallyassayedforitsabilitytoexpressatleastornativeapproachistobuildahighlydiverselibrary,onthebasisofcertainwell-expressingandprotein-LbindingKappalight-chaingenes(Holtetal.2008).Moreover,protein-Lhasaclearadvantageoverprotein-Aandprotein-G,ghtbeofmajorimportancewhenoneisforcedtousebovineserumasadditivetother,protein-Lhasnotbeenavailablefortheindustrial-scale6971989

24AnalysisandPurificationofAntibodyFragments303a3.53.02.52.01.51.00.501234mg/mLHD148HD147HD146HD149HD150bkDa755Time after inductionc1Protein-L234+

IgG-+ -Fig.24.2(a)Apilot-scaleprosequenceencodingtheB1-4domainshasbeenclonedintoapJB-vector(Slettaetal.2004)andtherecombinantprotein-Lwasexpressedintracellularinhigh-cell-density-cultivationasshownhereforfiducedprotein-Lwasextractedfromthecytoplasm,purified,andanalyzedbySDS-PAGE.(b)SDS-PAGEanalysisofthepurifiedprotein-L(c)CNBRactivated-sepharosebeadswereconjugatedwithout(À)andwithpolyclonalhumanIgG(þ)andincubatedwithprotein-Lpreparationswhichwerestoredfor1month,eitherat4