细菌双杂交

BacterialAdenylateCyclaseTwo-HybridSystem1BACTHSystemKitBacterialAdenylateCyclaseTwo-HybridSystemKitCatN°:EUK001FORRESEARCHUSEONLYNotforuseindiagnosticprocedures24,ruedesTuileriesBP68467460SOUFFELWEYERSHEIMCEDEXTél:0388180722Fax:[phone]e.mail:[e-mail]::Assayofβ-galactosidaseactivityNoticetoPurchaserTheBACTHsystemkitiscoveredbytheU.S.PatentNo.6,333,154.ResearchuseoftheBACTHsystembycommercialentitiesrequiresalicense;pleasecontact,Euromedexat(333)[e-mail]iswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarrantiesofanykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesofmerchantabilityorfitnessforaparticularpurpose,areprovidedbyEuromedex.Euromedexshallhavenoliabilityforanydirect,indirect,consequential,orincidentaldamagesarisingoutoftheuse,theresultsofuse,ortheinabilitytousethisproduct.BacterialAdenylateCyclaseTwo-HybridSystem3MaterialsSuppliedMaterialsAmountReferenceBACTHstrainsBTH101,LB/DMSOstock(reporterstrainforBACTHassay)1mlEUB001DHM1,LB/DMSOstock(reporterstrainforBACTHassay)1mlEUB002Storeat–80°CBACTHvectorspKNT25,supercoiled,10µg(0,5µg/µlinTEbuffer)20µlEUP-25NpKT25,supercoiled,10µg(0,5µg/µlinTEbuffer)20µlEUP-25CpUT18,supercoiled,10µg(0,5µg/µlinTEbuffer)20µlEUP-18NpUT18C,supercoiled,10µg(0,5µg/µlinTEbuffer)20µlEUP-18CStoreat–20°CControlplasmidspKT25-zip,supercoiled,1µg(0,05µg/µlinTEbuffer)20µlEUP-25ZpUT18C-zip,supercoiled,1µg(0,05µg/µlinTEbuffer)20µlEUP-18ZStoreat–20°CStorageconditions:Allplasmids:-20°CStrains:-80°CMaterialNotsuppliedMacConkeyagarbasemediumM63minimalmediumX-gal(5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside)IPTG(isopropyl-β-D-thiogalactopyranoside)ONPG(o-nitrophenol-β-galactoside)AmpicillinKanamycinMaltoseStandardE.coliK-12lacIqstrainsBacterialAdenylateCyclaseTwo-HybridSystem4PrincipleofBACTHsystemEuromedexbacterialtwo-hybrid(BACTH,forBacterialAdenylateCyclase-basedTwo-Hybrid”)systemisasimpleandfastapproachtodetectandcharacterizeprotein-proteininteractionsinvivo.Incontrasttoyeasttwo-hybridsystem,whichrequiresspecializedexpertise,BACTHoffersalltheadvantagesofworkingwithEscherichiacoli:readilyaccessiblemicrobiologyandmolecularbiologytechniques(plasmidpreparations,efficiencyoftransformation,PCR…).TheBACTHsystemhasbeendevelopedbythegroupofDr.D.LadantatthePasteurInstituteandisbasedontheinteraction-mediatedreconstitutionoftheadenylatecyclaseactivityinE.coli(1).Itexploitsthefactthatthecatalyticdomainofadenylatecyclase(CyaA)fromBordetellapertussis(2)consistsoftwocomplementaryfragments,T25andT18(Figure1A),thatarenotactivewhenphysicallyseparated(Figure1B).Whenthesetwofragmentsarefusedtointeractingpolypeptides,XandY,heterodimerizationofthesehybridproteinsresultsinfunctionalcomplementationbetweenT25andT18fragmentsand,therefore,cAMPsynthesis(Figure1C).CyclicAMPproducedbythereconstitutedchimericenzymebindstothecataboliteactivatorprotein,CAP.ThecAMP/CAPcomplexisapleiotropicregulatorofgenetranscriptioninE.coli.Itturnsontheexpressionofseveralresidentgenes,includinggenesofthelacandmaloperonsinvolvedinlactoseandmaltosecatabolism(Figure1D).Therefore,bacteriabecomeabletoutilizelactoseormaltoseastheuniquecarbonsourceandcanbeeasilydistinguishedonindicatororselectivemedia.Figure1:Principleofthebacterialtwo-hybridsystem.BacterialAdenylateCyclaseTwo-HybridSystem5OutlinedoftheprocedureDetectionofinvivointeractionsbetweentwoproteinsofinterestwiththeBACTHsystemrequirestheco-expressionoftheseproteinsasfusionswiththeT25andT18fragmentsinbacteriathatarelackingendogenousadenylatecyclaseactivity(E.colicya).Thisisachievedbyusingtwocompatiblevectors,oneexpressingtheT25fusion(pKT25orpKNT25)theotheroneexpressingtheT18fusion(pUT18orpUT18C).Thebacteriaareco-transformedwiththetworecombinantplasmidsandplatedoneitherindicatororselectivemediatorevealtheresultingCya+phenotype(Figure2).TheefficiencyofcomplementationbetweenthetwohybridproteinscanbefurtherquantifiedbymeasuringcAMPlevels(adirectmeasureofthereconstitutedadenylatecyclaseenzymaticactivity)orbyassayingtheβ-galactosidaseenzymaticactivitiesinbacterialextracts,aneasyandrobustassaythatiscorrelatedwithcAMPlevelsproducedinthecells,astheexpressionofβ-galactosidaseispositivelyregulatedbycAMP/CAP.ThehybridproteinsexpressedinE.coli,canalsobecharacterizedbyusingdiversebiochemicalapproaches,suchasimmunodetection,immunoprecipitation,copurification,etc....Figure2:Generalmethodologytoanalyzeprotein-proteininteractionswithBACTHsystemBacterialAdenylateCyclaseTwo-HybridSystem6AdvantagesoftheBACTHsystemTheBACTHsystempresentsseveralcharacteristicsthatmakesitaworthyalternativeand/orcomplementarytooltothetraditionalyeasttwo-hybridsystem:1-AstheBACTHassayiscarriedoutinE.coli,thescreeningandthecharacterizationofprotein