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

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1、Chapter24AnalysisandPurificationofAntibodyFragmentsUsingProteinA,ProteinG,andProteinLRemkoGriepandJohnMcDougall24.1IntroductionToday,monoclonalantibodies(mAbs)formthelargestcategoryofbiopharmaceu-ticalsinclinicaltrials,andtheirnumberisexpandingrapidly(DataMonitor2007a,b).Theantib

2、odiesorfunctionalantibodyfragmentsarebeingproducednotonlyinartificialproductionsystemssuchasmammaliancells,yeast,bacteria,andplantcellsbutalsointransgenicanimalssuchasgoats,sheep,andcows.Regardlessoftheproductionmethod,thequalitycontroldemandisthesameforallofthem.Hostcellproteins

3、,cellculturemediaadditives,DNA,andendotoxinshavetoberemovedfromthemAbpreparationtoallowtheproteinstobesafelyappliedforhumantherapy.Moreover,antibodyaggregates,clippedandlowmolecularweightspecies,shouldalsoberemoved.Severalproteinswithaninherentaffinityforimmunoglobulins(Ig)havebe

4、enisolatedfromvariousbacteria.Thesemoleculesincludeprotein-A,derivedfromStaphylococcusaureus(ForsgrenandSjo¨quist1966);protein-G,derivedfromagroup-CStreptococcus(Bjo¨rkandKronvall1984);andfinallyprotein-L,derivedfromPeptostreptococcusmagnus(A˚kerstro¨mandBjo¨rk1989;Housdenetal.20

5、03,2004).Theyallcontainrepetitive55–76aminoacidresidues(Fig.24.1)thatmediatetheactualIgbinding(Kasternetal.1992).Therecombinantprotein-Lcanbeproducedatayieldofupto3g/Linpilot-scalestudies.Ityieldsahighlypure,stable,andactiveprotein-Lfractionafterpurification,whichisbindingefficien

6、tlytomostofthehumanantibodiesoftheKappaisotype(Fig.24.2).Protein-GbindsnotonlytotheFc-regionbutalsototheCH1-domainofthehumanIgG1-isotype.Therefore,ithasabroaderapplicationcomparedtoprotein-A.Someacademicgroupshavealsoreportedtheuseofgeneticallyfusedprotein-LG(Kihlbergetal.1996;H

7、arrisonetal.2008)orprotein-AG(Eliassonetal.1988;R.Griep(*)andJ.McDougallAffitechAS,Gaustadalle´en21,Oslo3490,Norwaye-mail:r.griep@affitech.comR.KontermannandS.Du¨bel(eds.),AntibodyEngineeringVol.2,301DOI10.1007/978-3-642-01147-4_24,#Springer-VerlagBerlinHeidelberg2010302R.Griepand

8、J.McDougallAB1B2B3B4B5C1C2WSPSM1989174246318390