5-Investigation of Phage Display for the Directed Evolution of Enzymes

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1、DirectedMolecularEvolutionofProteins:orHowtoImproveEnzymesforBiocatalysis.EditedbySusanneBrakmannandKaiJohnssonCopyrightã2002Wiley-VCHVerlagGmbH&Co.KGaAISBNs:3-527-30423-1(Hardback);3-527-60064-7(Electronic)5InvestigationofPhageDisplayfortheDirectedEvolutionofEnzymesPatriceSoumillionan

2、dJacquesFastrez5.1IntroductionEngineeringenzymeswithnewandimprovedpropertiesorevennewactivitiesisbecomingagoalwithinreachofenzymologists.Infavorablecases,thiscanbedonebyrationaldesign.Frequently,however,ourunderstandingofthestructure±functionrelationshipremainstoolimitedforthisapproach

3、tobefruitful.Conse-quently,astrategyofdirectedevolutionisfrequentlybeingpursued.Librariesofmu-tantsarecreatedbyintroductionofrandommutations.Interestingmutantsarethensearchedeitherbyscreeningorselectiontechniques.Screeningmethodsinwhicheachcloneofthelibraryisanalyzedareprobablythemostp

4、opularbutsensitiveassayscanbedifficulttosetup,expensiveequipmentisusuallynecessaryandthemaximalsizeoflibrariesthatcanbescreenedisseveralordersofmagnitudelowerthanforselectionmethods.Invivoselectionisaverypowerfulandsimplemethodbutitsap-plicationispossibleonlywhentheinterestingphenotype

5、confersabiologicaladvan-tagetothehostcell[1].Moreover,invivoconditionsarerestrictedandpoorlycontrol-lable.Therefore,thereisagreatinterestindevelopinginvitroselectionstrategiesforengineeringenzymesendowedwithneworimprovedcatalyticproperties.Severalstrategieshavebeendevelopedwiththisaima

6、nd,inthefuture,phagedisplaycouldbecomeapowerfultechnologyforthedirectedevolutionofbiocatalysts.5.2ThePhageDisplayPhagedisplaytechnologyenablesinvitroselectionfromlibrariesofproteinsorpep-tidesdisplayedonthesurfaceoffilamentousbacteriophages.ItwasintroducedbyGeorgeP.Smithin1985[2]and,si

7、ncethen,severalthousandsresearchpapers805InvestigationofPhageDisplayfortheDirectedEvolutionofEnzymeshavereportedtheuseofthismethod(forareview,see[3]).Thepowerofthetechnologycomesfromthephysicallinkagebetweenapieceofgeneticinformationanditsex-pressionproductviaaphageparticle.Thisisach