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1、DirectedMolecularEvolutionofProteins:orHowtoImproveEnzymesforBiocatalysis.EditedbySusanneBrakmannandKaiJohnssonCopyright?2002Wiley-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
