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1、DirectedMolecularEvolutionofProteins:orHowtoImproveEnzymesforBiocatalysis.EditedbySusanneBrakmannandKaiJohnssonCopyrightã2002Wiley-VCHVerlagGmbH&Co.KGaAISBNs:3-527-30423-1(Hardback);3-527-60064-7(Electronic)7Yeastn-HybridSystemsforMolecularEvolutionBrianT.Carter
2、,HeningLin,andVirginiaW.Cornish7.1IntroductionIn1989FieldsandSongintroducedtheªYeastTwo-HybridAssayº,whichprovidesastraightforwardmethodfordetectingprotein-proteininteractionsinvivo[1].Therearethreesignificantadvantagesofthisinvivoassaythatledalmostimmediatelyto
3、itswidespreaduse:first,itistechnicallystraightforwardandcanbecarriedoutra-pidly;second,thesequenceofthetwointeractingproteinscanbereadoffdirectlyfromtheDNAsequenceoftheplasmidsencodingthem;andthird,itdoesnotdependontheidentityoftheinteractingproteinsandsoisgener
4、allyapplicable.Theyeasttwo-hybrid(Y2H)assaywasbasedontheobservationsthateukaryotictranscriptionalac-tivatorscanbedissectedintotwofunctionallyindependentdomains,aDNA-bindingdomain(DBD)andatranscriptionactivationdomain(AD),andthathybridtranscrip-tionalactivatorsca
5、nbegeneratedbymixingandmatchingthesetwodomains[2,3].ItseemsthattheDNA-bindingdomainonlyneedstobringtheactivationdomainintotheproximityofthetranscriptionstartsite,suggestingthatthelinkagebetweentheDBDandtheADcanbemanipulatedwithoutdisruptingactivity[4].Thus,theli
6、nkageintheY2Hassayisthenoncovalentbondbetweenthetwointeractingproteins.AsoutlinedinFig.7.1,theY2Hsystemconsistsoftwoproteinchimeras,andareportergenedownstreamfromthebindingsiteforthetranscriptionalactivator.Ifthetwoproteinsofinterest(XandY)interact,theyeffective
7、lydimerizetheDNA-bind-ingproteinchimera(DBD-X)andthetranscriptionactivationproteinchimera(AD-Y).DimerizationoftheDBDandthetranscriptionADhelpstorecruitthetranscriptionmachinerytoapromoteradjacenttothebindingsiteforthetranscriptionalactivator,therebyactivatingtra
8、nscriptionofthereportergene.Theassaywasdemonstratedinitiallyusingtwoyeastproteinsknowntobephysicallyassociatedinvivo[1].TheyeastSNF1protein,aserine-threonineproteinki
