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建立基于表面展示的高通量酵母双杂交筛选方法.docx

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建立基于表面展示的高通量酵母双杂交筛选方法 Title:DevelopmentofaHigh-throughputYeastTwo-hybridScreeningMethodBasedonSurfaceDisplay Abstract: Protein-proteininteractions(PPIs)playcrucialrolesinvariouscellularprocesses.Yeasttwo-hybrid(Y2H)isapowerfultechniquewidelyusedfortheinvestigationofPPIs.ThetraditionalY2Hmethod,relyingontheactivationofareportergeneintheyeastnucleus,haslimitationsintermsofsensitivityandthroughput.Inthisstudy,wepresentanovelhigh-throughputyeasttwo-hybridscreeningmethodbasedonsurfacedisplay.Thismethodallowsforthedirectvisualizationandquantificationofproteininteractions,enhancingbothsensitivityandthroughput.Wedemonstratetheutilityofthisapproachbyidentifyingseveralnovelproteininteractionsthatweremissedbypreviousscreeningmethods. Introduction: Protein-proteininteractions(PPIs)arecrucialfortheregulationofcellularprocesses,suchassignaltransduction,transcriptionalregulation,andcellcyclecontrol.TheidentificationandcharacterizationofPPIsareessentialforunderstandingtheunderlyingmechanismsoftheseprocesses.VariousmethodshavebeendevelopedtostudyPPIs,includingco-immunoprecipitation,affinitypurificationcoupledwithmassspectrometry,andyeasttwo-hybrid(Y2H)screening.Amongthesemethods,Y2HisparticularlypowerfulasitallowsforthesystematicinvestigationofPPIsinahigh-throughputmanner. TheclassicalY2Hsystemutilizestheinteraction-dependentreconstitutionofatranscriptionalactivatortodrivetheexpressionofareportergene,typicallyintheyeastnucleus.Althoughthistechniquehasbeenwidelyused,itisassociatedwithseverallimitations.Thenuclearcompartmentalizationcanrestrictinteractionsbetweencertainproteinpairs,leadingtofalse-negativeresults.Moreover,post-translationalmodificationsortheabsenceofnecessaryco-factorsintheyeastnucleusmayresultinfalse-positiveinteractions.Additionally,therelianceonreportergeneactivationinthenucleuslimitsthesensitivityandthroughputoftheclassicalY2Hmethod. Toovercometheselimitations,wehavedevelopedahigh-throughputyeasttwo-hybridscreeningmethodbasedonsurfacedisplay.Thisapproachinvolvesthe