Matchmaker GAL4 Two-Hybrid System 3 & Libraries User … Manual/PT3247...2007/04/03 · MEL1 is an endogenous GAL4-responsive gene.The lacZ reporter gene was introduced into PJ69-2A

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Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual

PT3247-1 (PR742219)Published 3 April 2007


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 2 Version No. PR742219

I. Introduction 4 II. Overview:AYeastTwo-HybridScreen 8

III. ListsofComponents 9

IV. AdditionalMaterialsRequired 10

V. YeastStrains&Phenotypes 11 A. YeastHostStrains 11 B. Phenotypes 11

VI. ControlVectors 13 A. PositiveControls 13 B. NegativeControl 13

VII. Matchmaker™GAL4cDNA&GenomicLibraries 14 A. LibraryConstruction 14 B. LibraryQualityControlInformation 15

VIII. PreparingforaTwo-HybridScreen 16 A. ConstructFusionGenes 16 B. ObtainorConstructandADFusionLibrary 16 C. VerifythatConstructsDoNotActivateReporterGenes 17 D. VerifyProteinExpression 17

IX. LibraryTransformation&ScreeningProtocols 18 A. TransformationScales 18 B. YeastTransformationProtocols 19 C. PlatingandScreeningTransformationMixtures 21 D. Calculations 23

X. Analysis&VerificationofPutativePositiveClones 24 A. RetestPhenotypes 24 B. IsolatePlasmidDNAfromYeast 24 C. SortColoniestoEliminateDuplicates 24 D. RescueAD/libraryPlasmidsviatransformationofE.coli 24 E. RetestProteinInteractionsinYeast 25 F. InvitroAnalysis 26 G. SequencingAD/LibraryInserts 27 H. InvivoAnalysis 27 I. AdditionalTwo-HybridTests 27

XI. TroubleshootingGuide 28

XII. References 30

XIII. RelatedProducts 32

AppendixA:Media&SolutionRecipes 33

AppendixB:LibraryTitering 34

AppendixC:PlasmidLibraryAmplification 35

TableofContents

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ListofTables

TableI. ListofAbbreviations 7TableII. MatchmakerYeastStrainGenotypes 11TableIII. MatchmakerYeastStrainPhenotypes 11TableIV. MatchmakerTwo-HybridSystem3Vectors 13TableV. ComparisonofTwo-HybridTransformationMethods 18TableVI. SetupforaTwo-HybridLibraryScreen 21

ListofFigures

Figure1. OverviewofMatchmakerTwo-HybridSystem3advancesandscreeningprocess 4Figure2. Thetwo-hybridprinciple 5Figure3. ReporterconstructsinyeaststrainsAH109andY187 5Figure4. Overviewofperformingayeasttwo-hybridscreen 8Figure5. ScreeninganADfusionlibraryusingstrainAH109 22Figure6. Strategiesforanalyzingandverifyingputativepositiveclones 25Figure7. Yeastmatingtoverifyproteininteractions 26

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I. Introduction

Principleofthetwo-hybridassay

InMatchmakerSystem3abaitgeneisexpressedasafusiontotheGAL4DNA-bindingdomain(DNA-BD),whileanothergeneorcDNAisexpressedasafusiontotheGAL4activationdomain(AD;Fields&Song,1989;Chienet al.,1991).Whenbaitandlibraryfusionproteinsinteract,theDNA-BDandADarebroughtintoproximity,thusactivatingtranscriptionoffourreportergenes(Figure2).Thistechnologycanbeusedtoidentifynovelproteininteractions,confirmsuspectedinteractions,anddefineinteractingdomains.Moreover,two-hybridtechnologyprovidesimmediateaccesstothegenesencodingtheinteractingproteins.

Sensitivein vivoassayYeasttwo-hybridsystemsprovideasensitivemethodfordetectingrelativelyweakandtransientproteininteractions.Suchinteractionsmaynotbebiochemicallydetectable,butmaybecriticalforproperfunctioningofcomplexbiologicalsystems(Guarente,1993;Estojaket al.,1995).Thesensitivity of Matchmaker GAL4Two-Hybrid System 3 is primarily attributable to high-foldamplificationofpositive signals in vivo (i.e., transcriptional, translational, andenzymatic). Inaddition,becausethetwo-hybridassayisperformedin vivo,theproteinsaremorelikelytobeintheirnativeconformations,whichmayleadtoincreasedsensitivityandaccuracyofdetection.

MatchmakerTwo-HybridSystem3isanadvancedGAL4-basedtwo-hybridsystemthatprovidesatranscriptionalassayfordetectingproteininteractionsin vivoinyeast.Youcanusethissystemtoscreenalibraryfornovelproteinsthatinteractwithaknownbaitprotein,ortotesttwopreviouslyclonedproteinsforaninteraction.MatchmakerTwo-HybridSystem3incorporatesmanyfeaturesthatreducetheincidenceoffalsepositiveresultsandallowyoutoquicklyidentifyandconfirmproteininteractions.KeyfeaturesofSystem3aredetailedinFigure1.

Figure1.OverviewofMatchmaker™System3advancesandscreeningprocess.

Library screening

α-gal or β-gal assay

Verification oftrue positives

Ade+, His+Colonies

Strain AH109

• Virtually eliminates false positives

• Allows stringency of selection to be varied

• Use simple α-gal or β-gal assay

Improved vectors

• Increased transformation efficiency

• High-level protein expression

• c-Myc and HA tags facilitate detection of fusion proteins

• Distinct bacterial selection markers AD—Ampr DNA-BD—Kanr

• High copy vectors • T7 promoter allows in vitro transcription and translation

Matchmaker™Co-IP Kit

Epitope-TaggedExpression Vectors

or MammalianMatchmaker™ Kit

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I. Introductioncontinued

Figure2.Thetwo-hybridprinciple.TheDNA-BDisaminoacids1–147oftheyeastGAL4protein,whichbindstotheGALUASupstreamofthereportergenes.TheADisaminoacids768–881oftheGAL4proteinandfunctionsasatranscriptionalactivator.

Thesensitivityofthetwo-hybridassaymeansthatitcanbeusedtopinpointspecificresiduescriticalforproteininteractionsandtoevaluateproteinvariantsfortherelativestrengthoftheirinteractions (Yang, et al.,1995).ThebindingdatareportedbyYanget al. (ibid.) lead themtosuggestthatproteininteractionswithdissociationconstants(Kd)above~70µMcanbedetectedusingaGAL4-basedtwo-hybridassay.

Newyeaststrainreducesfalsepositives

System3 features theyeast strainAH109,whichvirtuallyeliminates falsepositivesbyusingthreereporters—ADE2,HIS3,andMEL1 (or lacZ)—underthecontrolofdistinctGAL4upstreamactivatingsequences(UASs)andTATAboxes(Figure3).ThesepromotersyieldstrongandspecificresponsestoGAL4.Asaresult,twomajorclassesoffalsepositivesareeliminated:thosethatinteractdirectlywiththesequencesflankingtheGAL4bindingsiteandthosethatinteractwithtranscriptionfactorsboundtospecificTATAboxes.

TheADE2 reporteraloneprovidesstrongnutritionalselection:theoptionofusingHIS3selectionreducesthe incidenceof falsepositivesandallowsyoutocontrol thestringencyofselection(Jameset al.,1996).Furthermore,youhavetheoptionofusingeitherMEL1orlacZ,whichencodeα-galactosidaseandβ-galactosidase,respectively.MEL1isanendogenousgenefoundinseveralyeaststrains.Becauseα-galactosidaseisasecretedenzyme,itcanbeassayeddirectlyonX-α-Gal(Cat.No.630407)indicatorplates,whichemployblue/whitescreening.

Figure3.ReporterconstructsinyeaststrainsAH109andY187.StrainAH109isaderivativeofstrainPJ69-2AandincludestheADE2andHIS3markers(Jameset al.,1996).MEL1isanendogenousGAL4-responsivegene.ThelacZreportergenewasintroducedintoPJ69-2AtocreateAH109(A.Holtz,unpublished).TheHIS3, ADE2,andMEL1/lacZ reportergenesareunderthecontrolofthreecompletelyheterologousGAL4-responsiveUASandpromoterelements—GAL1,GAL2,andMEL1,respectively.StrainY187containsthelacZreportergeneundercontroloftheGAL1UAS.

GAL UAS minimal promoter Reporter gene

transcription

Bait protein

DNA-BD

Libraryprotein

AD

GAL1 UAS GAL1 TATA lacZ

Y1�7 Constructs

AH109 Constructs

GAL1 UAS GAL1 TATA HIS3

GAL2 UAS GAL2 TATA ADE2

MEL1 UAS MEL1 TATA lacZ

MEL1 UAS MEL1 TATA MEL1


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Optimizedvectorsfacilitatedownstreamconfirmation

TheMatchmakerSystem3DNA-BDandADfusionvectors,pGBKT7andpGADT7,weredesignedforhigh-levelproteinexpressionandtofacilitateconfirmationofproteininteractions.BaitandlibraryinsertsareexpressedasGAL4fusionswithc-Mycandhemagglutinin(HA)epitopetags,respectively.Theepitopetagseliminatetheneedtogeneratespecificantibodiestoeachnewproteinandallowconvenientidentificationofthefusionproteins.

System3vectorsalsoincludeT7promotersdownstreamoftheGAL4codingsequences.Thesepromotersallowyoutotranscribeandtranslatetheepitope-taggedfusionproteinsin vitro.TheMatchmakerCo-IPKit(Cat.No.630449)allowsyoutoconfirmproteininteractionsthroughanin vitrocoimmunoprecipitation.TheT7promoterisalsoaprimingsiteforDNAsequencing.

Finally,pGBKT7andpGADT7weredesignedtoexpressdifferentbacterialselectionmarkers—kanamycinandampicillin,respectively—tosimplifytheirindependentisolationinE. coli.PurifyingtheplasmidsfromE. coliallowstheisolationofADvectorsafteralibraryscreening.Eachvectoralsocontainsthehigh-copypUCoriginofreplicationtoensurehighyieldsfromplasmidpreparations.These features provide the high quality and quantity of DNA necessary for sequencing andcharacterizinginsertsbyPCRorrestrictiondigests.

Increasedtransformationefficiency

AnotherbenefitofSystem3isthatyeaststrainscarryingpGBKT7aretransformedmoreefficientlythanstrainscontainingotherDNA-BDvectors(Louretet al.,1997;Clontechniques,January1999).ThishighertransformationefficiencyfacilitatestheintroductionofADfusionlibrariesintoyeast,whichmaintainsthecomplexityofthelibraryandincreasestheprobabilityofdetectingnoveltwo-hybridproteininteractions.

Considerations

TheMatchmakerSystemshavebeenusedtoidentifymanydifferenttypesofproteininteractions,includingthosefromprokaryotes,yeast,plants,Drosophila,andmammals.However,allyeasttwo-hybridsystemshavepotentiallimitations:

• SomebaitproteinsmayhaveintrinsicDNA-bindingand/ortranscriptionalactivatingproperties;hence,deletionofcertainportionsofbaitproteinsmayberequiredtoeliminateunwantedactivitybeforetheproteinscanbeusedinatwo-hybridscreen(Bartelet al.,1993b).

• Somehybridproteinsmaynotbestablyexpressedinyeastorlocalizedtotheyeastnucleus.Forproteininteractionsthatnormallyoccuronthecellsurface,aphagedisplaysystemmaybeamoreappropriatechoice.However,thetwo-hybridsystemhasbeenusedtoidentifyextracellularproteininteractions(Ozenberger&Young,1995;Kuoet al.,1992).

• Insomecases,theDNA-BDorADfusionmoietymayoccludethenormalsiteofinteraction,ormayimpairtheproperfoldingofthehybridprotein,andthusinterferewiththeabilityofthetwohybridproteinstointeract(vanAelstet al.,1993).

• Theconditionsinyeastcellsmaynotallowtheproperfoldingorposttranslationalmodifications(suchasglycosylation)requiredforinteractionofsomemammalianproteins.Conversely,thedetectionofaspecificinteractionbetweenmammalianproteinsinayeastsystemdoesnotnecessarily indicatethatthereisacorrespondinginteractionintheproteins’nativeenvironment(Fields&Sternglanz,1994).

• SomeproteininteractionsmaynotbedetectableinaGAL4-basedtwo-hybridsystem,butmaybedetectableusingaLexA-basedsystemsuchastheMatchmakerLexATwo-HybridSystem(Cat.No.K1609-1;Gyuriset al.,1993;reviewedinGolemiset al.,1996;andMendelsohn&Brent,1994),andviceversa.Wecannotpredictwhichsystemwillgivethebestresultsforparticularproteininteractions.




table i. list of abbreviations

Two-HybridTerminology

AD/library AfusionoftheGAL4ADwithalibrarycDNA/protein.

DNA-BD/bait AfusionoftheGAL4DNA-BDwithabaitcDNA/protein.

YeastPhenotypes

Ade–,His–,Leu–,orTrp– Requiresadenine(Ade),histidine(His),leucine(Leu),ortryptophan (Trp)inthemediumtogrow;isauxotrophicforatleastoneofthese specificnutrients.

Ade+ ExpressestheADE2 reportergene;i.e.,doesnotrequireAdeinthe mediumtogrow.

His+ ExpressestheHIS3 reportergene;i.e.,doesnotrequireHisinthe mediumtogrow.

LacZ+ ExpressesthelacZreportergene;i.e.,ispositiveforβ-galactosidase activity.

Mel1+ ExpressestheMEL1reportergene;i.e.,ispositiveforα-galactosi daseactivity.

Miscellaneous

Ade2p Proteinencodedbytheyeast ADE2gene.

3-AT 3-amino-1,2,4-triazole;acompetitiveinhibitoroftheHis3protein.

CHX Cycloheximide

DO Dropout(supplementorsolution);amixtureofspecificaminoacids andnucleosidesusedtosupplementSDbasetomakeSDmedium; DOsolutionsaremissingoneormoreofthenutrientsrequiredby untransformedyeasttogrowonSDmedium.

His3p ProteinencodedbytheyeastHIS3 gene.

SDmedium MinimalSyntheticDropoutmedium;comprisedofanitrogenbase, acarbonsource(glucoseorgalactose),andaDOsupplement.

YPH YeastProtocolsHandbook(PT3024-1)

CompatibleMatchmaker™products

• Pretransformed Matchmaker™ cDNA Libraries provide a high-quality library previouslytransformedintoY187.SimplymatetoAH109thathasbeentransformedwithyourbait.

• Matchmaker™GAL4cDNALibrariesprovideaconvenientmeanstoquicklyscreenahigh-qualitypremadecDNAlibrary.

• pBridgeTMThree-HybridVector(Cat.No.630404)allowsyoutoinvestigateternaryproteincomplexes(Tirode et al.,1997).pBridgeallowsexpressionoftheDNA-BD/baitandathirdprotein.Thethirdproteinisonlyexpressedintheabsenceofmethionine.

• TheMatchmaker™Co-IPKit(Cat.No.630449)providesreagentsforquicklyandindependentlyconfirmingproteininteractionsthroughanin vitrocoimmunoprecipitation.

• Matchmaker™Antibodiesallowyoutoeasilydetectfusionproteins.SeeRelatedProductsfordetails.

•ThepCMV-Myc&pCMV-HAVectorSet(Cat.No.631604)allowsin vivocoimmunoprecipitationsinmammaliancells.

• TheMammalianMatchmaker™Two-HybridAssayKit(Cat.No.630301)isidealforconfirmingproteininteractionsinmammaliancells.

• X-α-Gal(Cat.No.630407)allowsyoutodetect α-galactosidaseactivity.


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II.Overview:AYeastTwo-HybridScreen

Figure4.Overviewofperformingayeasttwo-hybridscreen.TheappropriateUserManualsectionsareindicated.

Construct DNA/bait

Section VIII.A

Select Transformants

High Stringency: SD/–Ade/–His/–Leu/–Trp/X-α-GalMedium Stringency: SD/–His/–Leu/–Trp

Low Stringency: SD/–Leu/–TrpSection IX.C

(1 week) (1–3 weeks)

(5 days–2 week)

(3 days–1 week)

(3 hr)

(1 week–3 months)

Construct AD/target

Section VIII.A

Construct or Obtain AD/Library

Section VIII.B

Titer and Amplify Library

Appendix B & C

Test for Autonomous Activation

Section VIII.C

Transform AH109

Use sequential or simultaneous methodsSection IX.B

Verify Protein Interactions

Section X

(3 days–1 week)



III. ListsofComponents

A. Matchmaker™Two-HybridSystem3 Storeallyeaststrainsat–70°C.

StoresequencingprimersandplasmidDNAat–20°C.

• 50 µl pGBKT7CloningVector(0.1µg/µl)

• 50 µl pGADT7CloningVector(0.1µg/µl)

• 50 µl pGBKT7-53ControlVector(0.1µg/µl)

• 50 µl pGBKT7-LamControlVector(0.1µg/µl)

• 50 µl pGADT7-TControlVector(0.1µg/µl)

• 50 µl pCL1ControlVector(0.1µg/µl)

•0.5 ml AH109Saccharomyces cerevisiaeinYPDAmedium/25%glycerol.

•0.5 ml Y187Saccharomyces cerevisiaeinYPDmedium/25%glycerol.

• 40 µl T7SequencingPrimer(0.1µg/µl) 5'–TAATACGACTCACTATAGGGC–3'(21-mer)

• 40 µl 3'DNA-BDSequencingPrimer(0.1µg/µl) 5'–ATCATAAATCATAAGAAATTCGCC–3'(24-mer) • 40 µl 3'ADSequencingPrimer(0.1µg/µl) 5'–AGATGGTGCACGATGCACAG–3'(20-mer) • 10g –Leu/–TrpDropoutSupplement • 10 g –Ade/–His/–Leu/–TrpDropoutSupplement • 1 ml HerringTestesCarrierDNA,denatured • VectorInformationPackets(PT3248-5&PT3249-5)

B. Matchmaker™GAL4cDNA&GenomicLibraries Storeallcomponentsat–70°C.

Dividethelibraryculturesinto100µlaliquots,andstoreat–70°C.Avoidmultiplefreeze/thawcycles.

Retiterandamplifythelibrarybeforeuse(AppendicesBandC).

Theappropriatevectorinformationisprovidedintheaccompanyingvectorinformationpacket.

•2x1.0ml PlasmidLibraryCultureinLBbroth/25%glycerol

• 0.5 ml AH109Saccharomyces cerevisiaeinYPDAmedium/25%glycerol.

• 0.5 ml CG-1945Saccharomyces cerevisiaeinYPDmedium/25%glycerol.



IV.AdditionalMaterialsRequired

Thefollowingreagentsandmaterialsarenotsuppliedbutarerequired.Recipesforthesemateri-alsareprovidedinAppendixAandtheYPH.

•YPDAortheappropriateSDliquidmedium

•Sterile1XTE/LiAc(Prepareimmediatelypriortousefrom10Xstocks)

•TEbufferorsterile,distilledH2O

•Appropriatesteriletubesorflasksfortransformations.

•AppropriateSDagarplates Notes: • Preparetheselectionmediaandpourtherequirednumberofagarplatesinadvance. • AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hrpriortoplatinganytrans-

formationmixtures.Moisturedropletsontheagarsurfacecancauseunevenspreading.

• Yeastmaker™YeastTransformationSystem(Cat.No.630439)providesallthenecessaryreagentsforyeasttransformations.Note:

BoilthecarrierDNAfor20minandquicklycoolitonicejustpriortouse.

• SterilePEG/LiAcsolution(Prepareimmediatelypriortousefrom10Xstocks)

• 100%DMSO(Dimethylsulfoxide;SigmaCat.No.D-8779)

• 1XTEbuffer

• Sterile glass rod, bent pasteur pipette, or 5-mm glass beads for spreading cells onplates.

•65%Glycerol/MgSO4solutionforthelow-stringency procedure.

•X-α-Gal(Cat.No.630407)

•



V.YeastStrains&Phenotypes

ThissectionprovidesdetailedphenotypesoftheyeaststrainsincludedwithMatchmakerSystem3andMatchmakerGAL4cDNAandGenomicLibraries.Foradditionalinformationonthegrowthandmaintenanceofyeast,seetheYPH,ChapterIII.WealsorecommendGuthrie&Fink’sGuide to Yeast Genetics and Molecular Biology (1991)andHeslot&Gailardin’sMolecular Biology and Genetic Engineering of Yeasts (1992).

A. YeastHostStrains ThecompletegenotypesofAH109,Y187,andCG-1945areprovidedinTableII.Allstrainsare

gal4–andgal80–;thispreventsinterferenceofnativeregulatoryproteinswiththeregulatoryelementsinthetwo-hybridsystem.

1.UseAH109asthehoststrainifyouplantoscreenanAD/libraryusingHIS3,ADE2,andMEL1.

2.System3UsersOnly:UseY187asthehoststrainifyouplantotestforaninteractionbetweentwoknownproteinsusingthe lacZreporteronly. Inaddition,useY187asamatingpartnertoverifyproteininteractions.

3.LibraryUsersOnly:UseCG-1945asthehoststrainifyouplantoseparateDNA/baitandAD/library plasmids by cycloheximide counterselection. Alternatively, use pGBKT7toconstructyourbait.ThisDNA-BDvectorcontainsakanamycin resistancemarker;therefore,vectorscanbeseparatedinE. coli withoutcycloheximidecounterselection.

table ii. matchmaker™ yeast strain genotypes Strain Genotype References

AH109 MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, Jameset al.,1996; gal4Δ,gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, A.Holtz,unpublished GAL2UAS-GAL2TATA-ADE2, URA3 : : MEL1UAS-MEL1 TATA-lacZ

Y187 MATα,ura3-52,his3-200, ade2-101, trp1-901, Harperet al.,1993 leu2-3,112,gal4Δ,met–,gal80Δ, URA3 : : GAL1UAS-GAL1TATA-lacZ

CG-1945 MATa,ura3-52,his3-200, ade2-101,lys2-801, Feilotteret al.,1994; trp1-901, leu2-3,112,gal4-542,gal80-538,cyhr2, C.Giroux,pers.comm. LYS2 : : GAL1UAS-GAL1TATA-HIS3, URA3 : : GAL4 17-mers(x3)-CYC1TATA-lacZ B. Phenotypes 1.NutritionalRequirements Toverifyphenotypesandtobecomefamiliarwiththeyeaststrains,testthemforthe

phenotypesshowninTableIII.

a. Streakeachstrainontoadenine-supplementedYPD(YPDA)plates.Incubateat30°Cfor3–5days.Propagateadditionalculturesonlyfromisolatedcolonies.

Note:Thestockmayberefrozenseveraltimeswithoutsignificantlydecreasingviability.

b. Usingasterilelooportoothpick,streak3–4coloniesontotheindicatedSDselectionplates.

c. Incubateplatesat30°Cfor4–6days;yeaststrainsgrowmoreslowlyonSDselectionmediathanonYPDA.

d. SealstockplatewithParafilm,andstoreat4°C.

table iii. matchmaker™ yeast strain phenotypes Strain SD/–Ade SD/–Met SD/–Trp SD/–Leu SD/–His SD/–Ura YPDAYPD/CHX

AH109 – + – – – + + –

Y187 – – – – – + + –CG-1945 – + – – – + + +



V.YeastStrains&Phenotypescontinued

2.ColonyColorandSize a. Y187andCG-1945carrythe ade2-101mutation.Onmediumwithlowamountsof

adenine,thecolonieswillturnpinkafterafewdaysandmayturndarkerasthecolonyages.Thesecoloniesgrowto>2mmindiameter.However,small(



VI.ControlVectors

MatchmakerTwo-HybridSystem3providespositiveandnegativecontrolvectors.VectorinformationisprovidedinTableIV.

A. PositiveControls pCL1 encodes the full-length, wild-type GAL4 protein and provides a positive control for

α-galactosidaseand β-galactosidaseassays.

pGBKT7-53andpGADT7-TencodefusionsbetweentheGAL4DNA-BDandADandmurinep53andSV40largeT-antigen,respectively.p53andlargeT-antigeninteractinayeasttwo-hybridassay(Li&Fields,1993;Iwabuchiet al.,1993).

B. NegativeControl pGBKT7-LamencodesafusionoftheDNA-BDwithhumanlaminCandprovidesacontrolfor

afortuitousinteractionbetweenanunrelatedproteinandeitherthepGADT7-TcontroloryourAD/libraryplasmid.LaminCneitherformscomplexesnorinteractswithmostotherproteins(Bartelet al.,1993b;S.Fields,pers.comm.;Ye&Worman,1995).

table iv. matchmaker™ two-hybrid system 3 vectors

Fusion Epitopea Yeastselection Bacterialselection

CloningvectorspGBKT7 DNA/bait c-Myc TRP1 kanamycinpGADT7 AD/library HA LEU2 ampicillin

ControlvectorspCL1 GAL4 LEU2 ampicillinpGADT7-T AD/T-antigen HA LEU2 ampicillinpGBKT7-53 DNA-BD/p53 c-Myc TRP1 kanamycin

pGBKT7-Lam DNA-BD/laminC c-Myc TRP1 kanamycinaHA=hemagglutinin



YoucanusepremadeMatchmakerGAL4cDNAandGenomicLibrarieswithallMatchmakerGAL4-basedsystems.

A. LibraryConstruction cDNAlibrariesarepreparedusingamodifiedGubler&Hoffmanprocedure(1983).

cDNAPrimingMethods • Oligo(dT)primingeliminatesthesynthesisoflengthypoly(dT)regionsandensuresthat

full-lengthclonesand3'endswillbewell-representedinthelibrary(Chenchiket al.,1994;Borson,et al.,1992;Moqadam&Siebert,1994).

• Oligo(dT)+random-primingmayleadtoagreaterrepresentationofallportionsofthegene, includingamino-terminaland internaldomains,regardlessofmRNAsecondarystructure;randomprimingalsogeneratesawidersize-rangeofcDNA.

• Unidirectionallibrariesaremadewitholigo(dT)primersthathaveonevector-compatiblerestrictionenzymesite.Theothersiteisadded(withstickyends)bytheadaptorthatisligated to thecDNA.Thus,digestionwithone restrictionenzymeensures thecDNA'sproperorientationwhenligatedtoavectorthathasbeendigestedwiththeappropriatetwoenzymes.

AdaptorsandLinkers PleaserefertotheProductAnalysisCertificate(PAC)forinformationonthespecificadaptor

orlinkerusedintheconstructionofyourMatchmakerLibrary.

Notes: • TheopenreadingframeoftheinsertstartsatthecodonimmediatelyfollowingtheC-terminalcodon(a.a.881)

oftheGAL4AD,notwithintheadaptor.

• IfanEcoRIlinkerisused,thecDNAismethylatedtoprotectanyinternalEcoRIsites. • Ifanadaptorisusedintheconstructionofnondirectionallyclonedlibraries,nomethylationorrestrictionenzyme

digestionofthecDNAisrequired;therefore,anyinternalEcoRIsitespresentinthecDNAwillnotbecut. • Ifanadaptorisusedintheconstructionofunidirectionallyclonedlibraries,thecDNAismethylatedtoprotect

thealternativesite.

• IfthelibraryissynthesizedusingEcoRI/NotI/SalIadaptors,youmayexcisetheinsertsfromthevectorusingsiteswithintheadaptor.

cDNASizeFractionation Theadaptor-ligateddouble-strandedcDNAissize-fractionatedtoremoveunincorporated

primers,unligatedadaptors,andadaptordimers;thisprocessalsoremoveslow-molecularweight(

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B. LibraryQualityControlInformation ThefollowinginformationisprovidedonthePAC.Thesedatawereobtainedatthetimeof

libraryconstruction.

NumberofIndependentClones Thenumberof independentclones isestimatedbeforeamplification.Most librariesare

guaranteedtohaveatleast1x106independentclones.

LibraryTiter Librarytiterisdeterminedafteramplificationandmustbe>108cfu/mlforplasmidlibraries.

InsertSizeAnalysis Theinsertsizeof15randomlyselectedclonesisdeterminedbyPCRamplificationusing

insertscreeningprimers.

SequenceRepresentation Sequencerepresentationisevaluatedbycolonyhybridizationusingagene-specificprobe.

AllHumanMatchmakercDNALibrariesmustshowaminimum β-actinfrequencyof0.1%,andallothermammalianMatchmakercDNALibrariesmustshowaminimum β-actinfrequencyof0.05%.NonmammaliancDNAlibrariesarescreenedwithaubiquitouslyexpressedspecies-specificprobe.

Note:Thefrequencyofβ-actinpositiveclonesvariesamonglibrariesmadewithRNAfromdifferenttissuesandspecies.Afrequencyof>0.1%inahumancDNAlibrarysuggestsareasonablyhighprobabilityoffindingararetranscript(Hagenet al., 1988).FornonhumanmammaliancDNAlibraries,afrequencyof0.05%suggestsareasonablyhighprobabilityoffindingararemessage(Clontechobservations,unpublished).

PresenceofGenomicDNAorrRNASequencesincDNALibraries ThepurifiedpolyA+RNAusedtoconstructMatchmakercDNAlibrariesisnottreatedwith

DNase,duetopotentialdegradationbycontaminatingRNaseactivity.Therefore,thepolyA+preparationmayhave


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 1� Version No. PR742219

A. ConstructFusionGenes Thefollowingisabriefprotocolonconstructinggenefusions.Formoredetailedinformation,

see Sambrook et al. (1989).The orientation and reading frame of each fusion must bemaintainedinordertoexpressfusionproteins.

• Youcangenerateafusiongeneifcompatiblerestrictionsitesarepresentinthetestgenesandthecorrespondingvector. Ifnot,generatethegenefragmentbyPCRwithusefulrestrictionsitesincorporatedintotheprimers(Scharf,1990).ArestrictionsiteattheendofagenecanoftenbechangedintoadifferentsiteorputintoadifferentreadingframeusingaPCRprimerthatincorporatesthedesiredmutation.

• Ifyouareinvestigatingtwoknowngenes,useeithervector—unlessonehasanactivationorDNA-bindingactivitythatwouldinterferewiththeproperfunctioningofthetwo-hybridsystem.

1.Purifythegenefragment,whethergeneratedbyPCRorcutoutofaplasmid. Note:WerecommendtheNucleoSpinExtractionKit(Cat.No.635961)forrapidisolationofDNAfragments.

2.DigesttheDNA-BDorADvectorwiththeappropriaterestrictionenzyme(s),treatwithphosphatase,andpurify.

3.Ligatetheappropriatevectorandinsert.TransformligationmixturesintoE. coli. 4.Identifyinsert-containingplasmidsbyrestrictionanalysisorPCRusingtheMatchmaker

InsertScreeningAmplimerSet(Cat.No.630433). 5.UsetheSequencingPrimersincludedwithMatchmakerSystem3tochecktheorientation

andreadingframeofthejunctions.

B. ObtainorConstructanADFusionLibrary PremadeMatchmakercDNALibrariesandPretransformedcDNALibrariesfromavarietyof

tissuesandspeciesareavailablefromClontech.Alternatively,constructanADfusionlibraryinpGADT7usingeitherintronlessgenomicDNAorcDNAsuchthatatleast106differenthybridproteinswillbeexpressed(Ausubelet al.,1995).

Notes: • Two-hybridlibrariesareusuallyconstructedintheADvectorratherthantheDNA-BDvector.Fusingrandomproteins

toaDNA-BDwillproduceamuchlargerpercentageoffusionsthatfunctionasautonomoustranscriptionalactivators(Ma&Ptashne,1987).

• OtherGAL4ADvectorsarecompatiblewithMatchmakerTwo-HybridSystem3,providedtheycarrytheLEU2 nutritionalmarker.

1.Amplificationofpremadelibraries ObtainpremadelibrariesasE. coli transformants,notaspurifiedDNA.Amplifythelibraryto

produceenoughplasmidDNAtoscreenthelibraryinyeast.IfyouhaveobtainedaMatchmakercDNALibrary, followtheamplificationprotocolprovided inAppendixC. Ifyouhaveobtainedalibraryfromanothercommercialsource,followthemanufacturer’sinstructions.

2.ConstructionofcDNAlibraries UseanystandardmethodforgeneratingcDNA(Sambrooket al.,1989;Ausubelet al.,

1995).Fordetailedinformationregardingconstructionoftwo-hybridcDNAlibraries,seeVojteket al.(1993),Durfeeet al.(1993),Dalton&Triesman(1992),andLubanet al.(1992).

Besuretoreservea1.0-mlaliquotofyourlibrary,frozenin25%glycerol,sothatyoucangobackandamplifyitlater.

YoucanalsoconstructgenomicDNAlibrariesfromanorganismwhosegenomecontainsfeworno introns,suchasbacteriaoryeast.pGADT7containsauniqueBamHIsiteforconstructingaSau3AI-digestedgenomicDNAlibraryandauniqueCla Isite forconstructingalibraryaccordingtoJameset al.(1996).Note:AprocedurefortiteringaplasmidlibraryinE. coliisinAppendixB.

VIII.PreparingforaYeastTwo-HybridScreen



C. VerifythatConstructsDoNotActivateReporterGenes 1.IndependentlytransformyourDNA-BDandADfusionconstructsintostrainAH109.Assay

thetransformantsforMEL1 activationby selectingfortransformantsonSD/–Trp/X-α-GalandSD/–Leu/X-α-Gal,respectively.Performpositiveandnegativecontrolsinparallel(SeeSectionIX,TableVI).

Ifapartner isknownforyourDNA-BD/bait,use it tocheckwhetheran interaction isdetectablebeforeinvestinginalibrarysearch.

2.Ifthetransformantcoloniesarewhite,preparestockplatesandliquidculturesforfreezing. Iftransformantcoloniesareblue,seeSectionXIforpossiblesolutions.

D. VerifyProteinExpression 1.IndependentlytransformtheDNA-BDandADfusionconstructsintostrainAH109. 2.PrepareWesternblotsfromthetransformantsandprobetheblotswithantibodiesto

thec-MycandHAepitopetags(Cat.Nos.631206,631207)ortheGAL4DNA-BDandADMonoclonalAntibodies(Cat.Nos.630403,630402.Useuntransformedyeastasacontrol.SeetheYPH(SectionIV)forprotocolsonpreparingproteinextractsfromyeast.

Note:Usingpolyclonalantibodiesmayresultinmultiplecross-reactingbands.

Alternatively,usetheT7promotertotranscribeandtranslatetheepitope-taggedfusionproteinsin vitro.ConfirmproteintranscriptionandtranslationbycoimmunoprecipitationusingtheMatchmakerCo-IPKit(Cat.No.630449).

VIII.PreparingforaYeastTwo-HybridScreencontinued



IX. LibraryTransformation&ScreeningProtocols

Inthissection,weprovidedetailedprotocolsforpolyethyleneglycol/lithiumacetate(PEG/LiAc)-mediatedtransformationofyeast(Itoet al.,1983;Schiestl&Gietz,1989;Hillet al.,1991;Gietzet al., 1992).

The procedures described here are for library screens using strain AH109 only. If you choose to use strain CG-1945, follow the same general procedures and plate on SD/–His/–Leu/–Trp. DO NOT PLATE STRAIN CG-1945 ON MEDIA LACkING ADENINE; IT wILL NOT GROw.

A. TransformationScales Weprovideprotocolsforsmall-,large-,andlibrary-scaletransformations.TableVcompares

thetransformationmethods.

• Usesmall-scaletransformationsto: – VerifythattheDNA-BD/baitdoesnotautonomouslyactivatereportergenes – LookfortoxicityeffectsofDNA-BD/baitonhost – Performcontrolexperiments – TransformtheDNA-BD/baitforsequentialtransformations • Uselarge-scaletransformationswhenyouarelearningtwo-hybridscreening,orwhen

youdonothaveenoughDNAforalibrary-scaletransformation(TableV).Youcanperformeithersequentialorsimultaneoustransformations.

• In a sequential transformation, the DNA-BD/bait plasmid is introduced through asmall-scaletransformation;selectedtransformantsarethengrownupandtransformedwiththeADfusionlibrarythroughalarge-scaletransformation.AsequentialtransformationmaybepreferredbecauseitusessignificantlylessplasmidDNAthanasimultaneouscotransformation(TableV).

• Asimultaneouscotransformationisgenerallypreferredbecauseitiseasiertoperformthanasequentialtransformation—andbecauseoftheriskthatexpressionoftheDNA-BD/baitproteinmaybetoxictothecells.IftheDNA-BD/baitproteinistoxic,clonesarisingfromspontaneousdeletionsintheDNA-BD/baitplasmidwillhaveagrowthadvantageandwillaccumulateattheexpenseofclonescontainingintactplasmids.

• Library-scaletransformationsarepreferredwhenscreeninganAD/library. Inthecaseoflargeandlibraryscalesimultaneouscotransformations,youmustdetermine

thetransformationefficienciesofbothplasmidstogether,aswellasofeachtype ofplasmidindependently.ExamplecalculationsareshowninSectionIX.D.

table v. comparison of two-hybrid transformation methods Amount No.ofIndep. ofLimiting Amount Transformation Clones Transformation Plasmid ofCells Efficiency Amplified No.ofPlates

Library-scale 100–500µg 8ml 103–104 1x106 50x150-mmLarge-scaleSimultaneous 10–50µg 1.5ml 103–104 1x105 5x150-mmSequential 10–50µg 1.5ml 104–105 1x106 50x150-mmSmall-scale 0.1µg 1.5ml 105 na 1x100-mmna=notapplicableacfuperµgoftheADlibrary.b TotalapproximatenumberoftransformantsexpectedonSD/–Leu/–Trpselectionplatesassumingthat:1)theminimal

amountofplasmidwasused;2)thetransformedcellswereresuspendedinthevolumesrecommendedintheprotocol;3)200µloftransformedcellswerespreadoneachplate;and4)thetransformationefficiencieswereoptimal.



IX. LibraryTransformation&ScreeningProtocolscontinued

B. YeastTransformationProtocols Tipsforasuccessfultransformation •Usea1–3week-oldcolony(2–3mm)toinoculateeachliquidculture.Ifcoloniesonthe

stockplateare1.5).

5. Transferovernightculture(enoughtoproducean OD600=0.2–0.3)intothisvolumeofYPDA: 300ml 300ml 1L

6. Incubateat30°Cfor3hrwithshaking (230–270rpm).TheOD600willbe0.5±0.1.

7. Placecellsin50-mltubesandcentrifugeat 1,000xgfor5minatroomtemperature.

8. Discardthesupernatantandresuspendcell pelletsbyvortexinginthisvolumeofsterile TEorH2O: 25–50ml 25–50ml 500ml

9. Poolcellscentrifugeat1,000xgfor5min atroomtemperature.

10. Decantthesupernatant.

11. Resuspendthecellpelletinthisvolumeof freshlyprepared,sterile1XTE/LiAc: 1.5ml 1.5ml 8mlb

12. PreparePEG/LiAcsolution. 10ml 10ml 100mlaUseSD/–Trpwhenperformingthesecondtransformationinasequentialtransformationprotocol.bForlibrarycotransformationsonly:removetwo100-µlaliquotsofcompetentcellstoperformcontroltransformations

withpCL1,andpGBKT7-53+pGADT7-T.



TransformationScale

SMALL LARGE LIBRARY

13. Intheindicatedtube,addandmixthefollowing: 1.5ml 50ml 500ml •DNA-BD/baita 0.1µg 20–100µg 0.2–1.0mg

•AD/library 0.1µg 10–50µg 0.1–0.5mg

•HerringtestescarrierDNA 0.1mg 2mg 20mg

14. Addthisvolumeofyeastcompetentcells: 0.1 ml 1ml 8ml andmixwellbyvortexing.

15. AddthisvolumeofsterilePEG/LiAcsolution: 0.6ml 6ml 60ml andvortexathighspeedtomix.

16. Incubateat30°Cfor30minwith shaking(200rpm).

17.AddthisvolumeofDMSO: 70µl 700µl 7.0ml Mixwellbygentleinversionorswirling. Donotvortex.

18. Heatshockfor15minina42°Cwaterbath. Forlarge-andlibrary-scale,swirloccasionally tomix.

19. Chillcellsonicefor1–2min.

20. Centrifugecellsfor: 5sec 5min 5min

atroomtemperatureat: 14Krpm 1,000xg 1,000xg

21. Removethesupernatant.

22. Resuspendcellsinthisvolumeof1XTEb: 0.5ml 1.0mlor10mlc 10ml

23. ProceedtoSectionIX.Cforplating.

a Forsimultaneouscotransformation,werecommendamolarratioof2:1(DNA-BDvector:ADvector)foroptimaleffi-ciency.Forsequentialtransformations,addeithertheDNA-BDvectorconstructortheADvectorconstruct(notboth).

bIfusingthehighstringencyselectionmethod,resuspendcellsinYPDA.Themediawillaidtheyeastinrecoveryfromtheshockoftransformation,butwillnotadverselyaffectscreening.

cUse1.0mlforsimultaneouscotransformation.Use10mlforthesecondtransformationinasequentialtransformationprotocol.




C. PlatingandScreeningTransformationMixtures YoucanselectAH109transformantsusinghigh-,medium-,orlow-stringencymedia(Figure

5).Lessstringentscreensincreasethenumberoffalsepositives,whilemorestringentscreensmayresultinfalsenegatives.

• High-stringency:PlatetransformationsonSD/–Ade/–His/–Leu/–Trp/X-α-GalmediumtoscreenforADE2, HIS3, and MEL1 expression.Thisscreenvirtuallyeliminatesfalsepositiveinteractions;however,low-affinityproteininteractionsmaybemissed.

• Medium-stringency:PlatelibrarytransformationsonSD/–His/–Leu/–TrpmediumtoscreenforexpressionofHIS3.Plantoscreenatleast1.5–3timesthenumberofindependentclonesinthelibrary.Subsequently,replicaplateHis+coloniesontoSD/–Ade/–His/–Leu/–Trp/X-α-GalmediumtoscreenforADE2andMEL1expression.

• Low-stringency:Performthisscreenifyouarehavingtroublepickinguppositiveclonesorifyoususpectthatyourbaitproteininteractsveryweaklyortransientlywithotherproteins.

PlateoutlibrarytransformationsonSD/–Leu/–TrpmediumtoselecttheDNA-BDandADvectors.Thisselectionstepprovidesaninitialphaseofgrowththatmaximizesplasmidcopynumber,whichresultsinhigherlevelsoffusionprotein.This,inturn,improvesthechancesofdetectingADfusionproteinsthatinteractweaklyortransientlywiththebait.

Thisscreentypicallyresultsinupto1,000candidatecolonies.Therefore,youmustoptimizethe3-ATconcentrationneededtocontrolbackgroundgrowth.Furthermore,alowstringencyscreenmayresultinapopulationpreferenceforclonesexhibitingstrongeractivationoftheHIS3 reporter,andextrastepsmayberequiredtosorttheclonesintogroupsbeforeyouproceedwithfurtheranalysis.

table vi. set up for a two-hybrid library screen

Vectors Scalea SDMinimalMedium Amountto Phenotype Plate(µl)Mel1/LacZHis/AdeControlpCL1 S –Leu 100 Blue +pGADT7-T S –Leu 100 White –+pGBKT7-53 S –Trp 100 White – S –Leu/–Trp 200 Blue + S –Ade/–His/–Leu/–Trp/X-α-Galb 200 Blue +pGADT7-T S –Leu 100 White –+pGBKT7-Lam S –Trp 100 White – S –Leu/–Trp 200 White – S –Ade/–His/–Leu/–Trp/X-α-Galb 200 NoGrowth –

ExperimentalDNA-BD/bait L/Lib –Leuc 100 White –+ADlibrary L/Lib –Trpc 100 White – L/Lib Low:–Leu/–Trpd,e,f 100 White – L/Lib Medium:–His/–Leu/–Trpb,e 200 White/Blue–/+ L/Lib High:–Ade/–His/–Leu/–Trp/X-α-Galb 200 White/Blue–/+aS=smallscale;L=largescale;Lib=libraryscale.bIfnecessary,seeSectionV.B.5forguidelinesonhowmuch3-ATtoadd.cTotestthetransformationefficiencyofeachplasmid,dilute1µlofthetransformationwith100µlofH2O.Spread1µl

onto100-mmSD/–LeuandSD/–Trpplates.dTotestthecotransformationefficiencyspread100µlofa1:1,000,1:100,and1:10dilutiononto100-mmSD/–Leu/–Trp

plates.e Plateatleast1.5–3timesthenumberofindependentcolonies.f Ina low-stringency libraryscreen,use three–Hisselectionplates:onewith theoptimalconcentrationof3-AT;one

witha10–15-mMhigher3-ATconcentrationtocontrolforbackgroundgrowthdue;andonewitha5-mMlower3-ATconcentrationforimprovedgrowthofweakpositives.





Figure5.ScreeninganADfusionlibraryusingstrainAH109.Usethestringencyofyourchoicetoscreenforinteractingproteins.Note:highstringencyselectionsresultinfewercolonies,andreducethenumberoffalsepositives.However,weakinteractionsmaybemissed.

Plate culture on SD/–Leu/–Trp to select all cotransformants

Colony growth and blue color indicates an interaction between the two-hybrid proteins

Scrape colonies and make a glycerol stock

(Nearly confluent growth)

Cotransform AH109

DNA-BD/baitMarker: TRP1

AD/fusion libraryMarker: LEU2

Plate culture on SD/–His/–Leu/–Trp

Plate culture onSD/–Ade/–His/–Leu/–Trp/X-α-gal

Medium-stringency

Replica plate to SD/–Ade/–His/–Leu/–Trp/X-α-gal

or

Low-stringency

Plate culture on SD/–Ade/–His/–Leu/–Trp/X-α-gal

High-stringency

1.PlatetransformationmixturesasindicatedinTableV.Platesmall-scaletransformationson100-mmplatesandlarge-andlibrary-scaletransformationson150-mmplates.Withanewbaitandlibrarycombination,predictingtheoptimalmethodisdifficult.Therefore,plateathirdofthetransformationonlow-,medium-,andhigh-stringencyplates.

2.Incubateplatesupside-downat30°Cuntilcoloniesappear. 3.IfscreeninganAD/library,calculatethetransformationefficiencyandestimatethenum-

berofclonesscreened,asdescribedinSectionIX.D.




4.LOW-STRINGENCYPROTOCOLONLY.Harvestthelibrarytransformantsasfollows: a. Chillplatesat4°Cfor3–4hr. b. Add1–5mlofTEbuffer(pH7.0)toeachplate.Carefullyscrapethecoloniesintothe

liquidusingaheat-bent,sterilePasteurpipette.Combineallliquidsinasterile50-mltube,andvortextoresuspendthecells.

Note:Ifthecombinedvolumeistoolarge,reduceitasfollows:centrifugefor5minat1,000xg,removeallbut25–50mlofthesupernatant,andvortextoresuspendthecells.

c. Createaglycerolstockbyaddinganequalvolumeofsterile65%glycerol/MgSO4solution. d. Divideinto1-mlaliquots,andstoreat4°Cforaweekorat–80°Cupto1yr. e. TitertheglycerolstockonSD/–Leu/–Trp(AppendixB).Incubateplatesat30°Cfor

3–4daysoruntilcoloniesareeasytocount.Calculatethecfu/µloflibrary. f. Platetheamplifiedyeastcotransformantsathighdensityoneither: • High-stringencyplates:SD/–Ade/–His/–Leu/–Trp/X-α-gal • Medium-stringencyplates:SD/–His/–Leu/–Trp To compensate for possible errors in the amplified library titer, plate 0.5 x 106

cfuonsomeplatesand2x106cfuonothers.Also,plateappropriatecontrolsforcomparison.

Note: If you plate on medium-stringency plates, you must replica plate to high-stringency plates toeliminatefalsepositives.

g. Incubateplatesupside-downat30°Cuntilcoloniesappear. 5.ChooseAde+/His+/Mel1+coloniesforfurtheranalysis.

Note:After2–3days,someAde+/His+colonieswillbevisibleonthehigh-stringencyplates;however,incubateplatesfor5–10daystoallowweakpositivestogrow.Ignoresmall,palecoloniesthatappearafter2daysbutnevergrowto>2mmindiameter.TrueHis+coloniesarerobustandcangrowto>2mm.Ade+colonieswillremainwhitetopalepink;Ade–colonieswillgraduallyturnreddish-brownandstopgrowing.StrongerADE2expressionwillbewhite,whileweakerexpressionwillbeprogressivelymorered.

6.[Option]Performaβ-galactosidasefilterassay(YPH).

D. Calculations 1.CotransformationEfficiency. Countcolonies(cfu)growingontheSD/–Leu/–Trpdilutionplatethathasbetween30–300cfu:

cfuxtotalsuspensionvol.(µl) =cfu/µgDNA Vol.plated(µl)xdilutionfactorxµgDNAused* *Inacotransformation,thisistheamountoflimiting plasmid,notthetotalamountofDNA.

2.NumberofClonesScreened. cfu/µgxµgoflibraryplasmidused=No.ofclonesscreened Examplecalculation: • 100coloniesgrewonthe1:100dilutiontransformationefficiencycontrolplate(dilution

factor=0.01) •resuspensionvolume=10ml •amountoflibraryplasmidused=100µg

100cfu x (10mlx103µl/ml) =1x104cfu/µg 100µlx0.01x100µg

•1x104cfux100μg=1x106clonesscreened.

3.AmountofDNAtoUse.

Ifyouscreened



X.Analysis&VerificationofPutativePositiveClones

Thissectionprovidesprotocolsforverifyingproteininteractions.Figure6providesadetailedoverview.

A. RetestPhenotypes TheinitiallibrarycotransformantsmaycontainmorethanoneAD/libraryplasmid,which

cancomplicatetheanalysisofputativepositiveclones. 1.RestreakpositivecoloniesonSD/–Leu/–Trp/X-α-Galplates2–3timestoallowlossofsome

oftheAD/libraryplasmidswhilemaintainingselectivepressureonboththeDNA-BDandADvectors.Incubateplatesat30°Cfor4–6days.Amixtureofwhiteandbluecoloniesindicatessegregation.

2.Replicaplateortransferwell-isolatedcoloniestoSD/–Ade/–His/–Leu/–Trp/X-α-Galplatestoverifythattheymaintainthecorrectphenotype.

3.CollecttherestreakedandretestedAde+/His+/Mel1+coloniesonSD/–Ade/–His/–Leu/–Trpmasterplatesinagridfashion.Incubateplatesat30°Cfor4–6days.Aftercolonieshavegrown,sealplateswithParafilm,andstoreat4°Cforupto4weeks.

B. IsolatePlasmidDNAfromYeast TheYeastmakerYeast Plasmid Isolation Kit (Cat. No. 630441) provides the reagents and

aprotocolforisolatingplasmidDNAfromyeast.TheseproceduresprovideplasmidDNAsuitable for PCR and E. coli transformations.A similar protocol is provided in theYPH.Note:theplasmidDNAisolatedfromeachpositiveyeastcolonywillbeamixtureoftheDNA-BD/baitplasmidandatleastonetypeofAD/libraryplasmid.

Alternatively,youmaywishtotrythedirecttransferofplasmidDNAfromyeasttoE. coli byelectroporation(Marcil&Higgins,1992).

Note:Forthismethod,thetransformationefficiencyofcompetentE. colicellsmustbe>109cfu/mg.

C. SortColoniestoEliminateDuplicates 1.AmplifyAD/libraryinsertsbyPCRandcharacterizePCRproductsbydigestingwitha

frequent-cutterrestrictionenzyme,suchasAlu IorHae III.Analyzefragmentsizesbyagarosegelelectrophoresis;also,runasampleoftheuncutamplifiedinserttocheckformultipleAD/libraryplasmids.Prepareanewmasterplatewitharepresentativeclonefromeachgroup.Ifyouaresatisfiedwiththenumberofuniqueclones,proceedtoStep3.

Notes:ToamplifyAD/libraryinserts,werecommendtheMatchmakerADLDInsertScreeningAmplimerSet(Cat.No.630433)andtheAdvantage2PCRKit(Cat.Nos.639206,639207).

2.IfahighpercentageofthecoloniesappeartocontainthesameAD/libraryinsert,expandyourPCRanalysistoanotherbatchof50colonies.

Alternatively,eliminatetheabundantclonesbyperformingyeastcolonyhybridizationoneachmasterplate.Useavector-freeoligonucleotideprobedesignedfromthesequenceofthemostabundantinsert.Transferarepresentativeofeachtypeofinserttoanewmasterplate.

CAUTION:SomepositivecoloniesmaycontainmultipleAD/libraryplasmids—evenifthecolonyhasbeenrestreakedtwice.Therefore,ifapositivecolonyappearstohavemultipleAD/libraryplasmids,donotimmediatelyeliminatethosethatcontaintheabundantinsert.

3.Prepareaglycerolstockofeachuniquetype,andstorealiquotsat–80°C.

D. RescueAD/LibraryPlasmidsviaTransformationofE. coli. 1.LibraryUsers: • ForstrainCG-1945,usecycloheximide(CHX)counterselectiontoobtaincoloniesthathave

losttheDNA-BDandretainedtheAD.RefertotheYPH(ChapterIX)forthisprocedure. • IfyoutransformedstrainAH109anddidnotuseDNA-BDandADvectorswithdifferent

antibiotic markers, transform KC8 E. coli cells and plate on M9 medium lackingleucine.KC8cellshaveadefectin leuBthatcanbecomplementedbyyeast LEU2.

System 3 Users or Libraries User with a pGBKT7/bait: Transform the yeast-purifiedplasmid DNA into E. coli.To select for transformants containing only theAD/libraryplasmid,plateonLBmediumcontainingampicillin.

2.ToverifythatyouhaveobtainedthesameAD/libraryplasmid,amplifyinsertsbyPCR.Thendigest the fragmentwithAlu IorHae III, and runasmall sampleonan3–4%agarose/EtBrgel.ComparethePCRproductgelprofilesfromE. coliandyeast.



X.Analysis&VerificationofPutativePositiveClonescontinued

Figure6.Strategiesforanalyzingandverifyingputativepositiveclones.IfthelibraryscreenwasperformedusingstrainCG-1945,seetheYPHSectionIXfordetailsonhowtosegregatetheDNA-BD/baitandAD/libraryplasmids.

E. RetestProteinInteractionsinYeast Youcanretestproteininteractionsinyeastbyeithercotransformationoryeastmating.A

realinteractionwillbehavelikethecontrolsinTableVI. 1.Cotransformation a. Using thesmall scale transformationprocedure, transform theDNA-BD/baitand

AD/libraryplasmidsintoAH109. b. PlateonSD/–Ade/–His/–Leu/–Trp/X-α-gal. c.Incubateplatesat30°Cuntilcoloniesappear.

Confirm Interaction in Yeast

Cotransform DNA-BD/bait and AD/library plasmids into AH109

orPerform yeast matings

In vitro coimmunoprecipitationusing Matchmaker Co-IP Kit

(Cat. No. 630449)

Retest Ade+/His+/Mel1+(LacZ+) phenotype

Eliminate colonies bearing the same AD/library plasmid by

a) PCR or

b) Colony Hybridization

Isolate plasmids from yeast

Transform plasmids into E. coli and purify DNA

Confirm protein interactionsin mammalian cells

Mammalian Two-HybridAssay Kit

(Cat. No. 630301)

In vivo Co-IP usingpCMV-Myc & pCMV-HA

(Cat. No. 631604)

Additional Two-Hybrid Tests- Switch Vectors- Frameshift Mutations- Site-specific mutations/deletions

Sequence cDNA inserts




Figure7.Yeastmatingtoverifyproteininteractions.

(1) DNA-BD (2) DNA-BD/bait (3) DNA-BD/laminC

Master plate with candidate clones1. Transform AH109 with: a) AD/library b) AD vector

Plate on SD/–Leu

2. Inoculate 0.5-ml YPD cultures.

4. Plate on SD/–Leu/–Trp

3. Mate to Y187 transformed with:

5. Replica plate or streak onto SD/–Ade/–His/–Leu/–Trp/X-α-Gal

(4) DNA-BD (�) DNA-BD/bait

AH109 [AD vector]

2.YeastMating Yeastmatingisaconvenientmethodofintroducingtwoplasmidsintothesamehost

cells(Finley&Brent,1994;Harperet al.,1993).IfyouhavemanyAde+,His+,Mel1+/LacZ+positiveclonestoanalyze,itwillbemoreconvenienttohandletheclonesinbatchesof10orsoeach.

a. TransformAH109withtheAD/libraryandADplasmid,andselectonSD/–Leu. b. TransformY187(orasuitableMatαstrain)withthefollowingthreeplasmids,and

selectonSD/–Trpplates: i. DNA-BD ii. DNA-BD/bait iii. pGBKT7-Lam c. ForeachcandidateAD/libraryplasmidtobetested,setuptheyeastmatingsindicated

inFigure7usingtheTrp+andLeu+transformantsobtainedinStepsa&babove. d. RefertotheYPH,ChapterIXformatingprocedures.Toselectfordiploids,spread

matingmixturesonSD/–Leu/–Trpplatesasdirected. e. Streakorreplica-platetoSD/–Ade/–His/–Leu/–Trp/X-α-gal.TruepositivesareAD/library

clones exhibiting reporter gene expression only when theAD/library plasmid isintroducedbymatingwiththeplasmidencodingtheDNA-BD/baitprotein.Discardanyβ-galactosidase-positivecoloniescontainingtheAD/libraryplasmidalone.

F. In vitro Analysis

TheMatchmakerCo-IPKit(Cat.No.630449)allowsyoutoconfirmproteininteractionsquicklyandindependentlyviaan in vitrocoimmunoprecipitation.TheCo-IPKitworkswithallGAL4-basedMatchmakerSystemandLibraryvectors.BecauseSystem3vectorsalreadycontainT7promotersandepitopetags,youcanusethemdirectlyinin vitro transcription/translationreactions.ForallotherGAL4-basedvectors,youmustfirstusetheCo-IPPrimerstoamplifyinsertsinordertoincorporatetheT7promotersandepitopetags.TheCo-IPKitalsoprovidesc-MycandHAantibodiesforprecipitatinginteractingproteins.

1.Transcribeandtranslatetheepitope-taggedfusionproteins in vitrousingtheT7promotersintheADandDNA-BDvectors.Note:theT7promoterislocateddownstreamoftheGAL4codingsequence;hence,theGAL4domainsarenottranscribed.




2.Coimmunoprecipitatetheepitope-taggedfusionproteinsusingc-MycandHAantibodies(Durfeeet al.,1993;Zhanget al.,1993).

If the fusion proteins do not coimmunoprecipitate, use other means to confirm theinteraction. Note: protein interactions with weak affinities may escape detection bycoimmunoprecipitation.SeePhizichy&Fields(1995)fordetailsonmoresensitivedetectionmethods.Furthermore,theADfusionproteinsmaypotentiallynotbein-framewiththeepitopetag. See Section X.G. 2–5 for further recommendations.

G. SequenceAD/LibraryInserts UseonlyDNAisolatedfromE. coli. 1.SequenceinsertsinthepositiveAD/libraryplasmidsusingthe3'ADSequencingPrimer

andT7SequencingPrimerprovidedwithSystem3.Verifythepresenceofanopenreadingframe(ORF)fusedtotheGAL4ADsequence,andcomparethesequencetothoseinGenBank,EMBL,orotherdatabases.

2.If yoursequencing results revealapeptide



XI.TroubleshootingGuide

DNA-BD/baitactivatesreportergenes

Excessivebackground

Lowtransformationefficiency

Iftwotestproteinsarebeingassayed,switch from the DNA-BD to theADvectorandviceversa.

Remove the activating domain bycreatingspecificdeletionswithinthegene.Retestthedeletionconstructsforactivation.Attheaminoacidlevel,anetnegativechargeper10aminoacids is a minimal AD. Note thatsuchdeletionsmayalsoeliminateapotentiallyinteractingdomain.

Remake SD/–Ade/–His/–Leu/–Trp/X-α-Galmedium.Add theappropriateamountof3-AT(SectionV.B.5).

UsewaterorTE.

Remake media, test with controltransformations.

Switchtosequentialtransformation.

Repeat the experiment using moreof the plasmid that had the lowtransformationefficiency.

Check the purity of the DNA and,if necessary, repurify it by ethanolprecipitation.

If you are not already doing so,we strongly recommend using thepretestedandoptimizedYeastmakerCarrier DNA, which is availableseparately(Cat.No.630440),oraspartoftheYeastmakerYeastTransformationSystem(Cat.No.630439).

Repeatthetransformation,thistimeincluding a “recovery” period aftertheheatshock.Toprovidearecoveryperiod, perform the simultaneouscotransformation as described(SectionIX.B),butaddthefollowingstepsafterStepB.21:22. Resuspendcellsin1.0LofYPDA

medium for a library-scale,and 100 ml for a large-scale,transformation.

23. Incubate cells for 1 hr at 30°Cwithshakingat230rpm.

24. Pellet cells by centrifuging at1,000 x g for 5 min at roomt e m p e r a t u r e . R e m o v es u p e r n a t a n t . P r o c e e dtoStepB.22.

Thebaitproteinhasatranscriptionalactivationdomain.Thisisespeciallylikely if the bait protein is atranscription factor (Ma & Ptashne,1987; Ruden et al., 1991; Ruden,1992). Acidic amphipathic domainsare often responsible for unwantedtranscriptional activation (Ruden et al.,1991;Ruden,1992).

Impropermediapreparation.

ResuspensionoftransformedcellsinYPDAistoorich.

Impropermediapreparation.

A problem with simultaneouscotrans-formation, even though thetransformation with the AD libraryplasmidsalonegaveatransformationefficiencyof≥5x104cfu/µgandwiththe bait plasmid alone gave ≥105cfu/µg.

The AD library vector gave atransformationefficiencyof



XI.TroubleshootingGuidecontinued

Comparegrowthcurvesofhoststrainwith DNA-BD vector and DNA-BD/bait. If bait is toxic, use sequentialtransformations or switch to a lowexpressingDNA-BDvector.

Truncationofoneofthehybridproteinsmay alleviate the toxicity and stillallowtheinteractiontooccur.Tryusingvectors that express lower levels ofthefusionproteins,suchaspGBT9(aDNA-BDvector),andpGAD424,pGADGL,orpGAD10(ADvectors)(Holtz&Zhu,1995).

See previous tip on improvingtransformationefficiency.

Constructhybridscontainingdifferentdomains of the bait protein. Forexample, to study proteins thatnormallydonotlocalizetothenucleus,it may be necessary to generatemutantformsoftheproteinthatcanbe transported across the nuclearmembrane.

UsetheMatchmakerLexATwo-HybridSystem(Cat.No.K1609-1).

RefertoSectionXformethodstoverifyprotein interactions; see Bartel et al. (1993a) for further discussion of falsepositives.

In sequential transformation, ADtransforms poorly; even as emptyvector.Baitprotein ismildlytoxicorinhibitingtotransformation

High-levelexpressionofoneorbothof thehybridproteins is toxic to thecell;therefore,transformantswillnotgroworwillgrowveryslowly.Forthisreason,werecommendthatyoucheckfor cell toxicity before performing alibraryscreen(SectionVIII.D).

The transformationefficiencyofoneorbothplasmidsistoolow.Youmaynotbescreeningasufficientnumberof library cotransformants.This canbecritical,especiallyiftheinteractingtarget protein is encoded by a raretranscriptinthesourcetissue.

If one of the following situations isoccurring, it may interfere with theability of the two hybrid proteinsto interact: (1) the hybrid proteinsarenot stablyexpressed in thehostcell; (2) the fused GAL4 domainsoccludethesiteofinteraction;(3)thehybrid protein folds improperly; or(4) the hybrid protein cannot belocalizedtotheyeastnucleus.(SeevanAelstet al.[1993]foroneexample.)

Some types of protein interactionsmaynotbedetectableinaGAL4-basedsystem.

Some protein interactions are notdetectableusinganytypeoftwo-hybridassay.

A rare category of false positives inwhich anAD/library hybrid activatestranscriptioninappropriately.

Failuretodetectknownproteininteractions

AD/libraryplasmidactivatesallthreereportersindependentoftheDNA-BD/bait

Problem Cause Solution



XII.References

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Aho,S.,Arffman,A.,Pummi,T.&Uitto,J. (1997)Anovelreportergene MEL1 for theyeast two-hybridsystem.Anal. Biochem.253:270–272.

Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.&Struhl,K.(1995)CurrentProtocolsinMolecularBiology(JohnWiley&Sons).

Bartel,P.L.,Chien,C.-T.,Sternglanz,R.&Fields,S.(1993a)Usingthetwo-hybridsystemtodetectprotein-proteininterac-tions.InCellular Interactions in Development: A Practical Approach.,ed.Hartley,D.A.(OxfordUniversityPress,Oxford)pp153–179.

Bartel,P.L,Chien,C.-T.,Sternglanz,R.&Fields,S.(1993b)Eliminationoffalsepositivesthatariseinusingthetwo-hybridsystem.BioTechniques 14:920–924.

Bendixen,C.,Gangloff,S.&Rothstein,R.(1994)Ayeastmating-selectionschemefordetectionofprotein-proteininterac-tions.Nucleic Acids Res.22:1778–1779.

Borson,N.D.,Sato,W.L.&Drewes,L.R.(1992)Alock-dockingoligo(dT)primerfor5’and3’RACEPCR.PCR Methods Appl. 2:144–148.

Chenchik,A.,Diatchenko,L.,Chang,C.&Kuchibhatla,S.(1994).GreatLengthscDNASynthesisKitforhighyieldsoffull-lengthcDNA.ClontechniquesIX(1):9–12.

Chien,C.T.,Bartel,P.L.,Sternglanz,R.&Fields,S.(1991)Thetwo-hybridsystem:Amethodtoidentifyandclonegenesforproteinsthatinteractwithaproteinofinterest.Proc. Nat. Acad. Sci. USA 88:9578–9582.

Dalton,S.&Treisman,R.(1992)CharacterizationofSAP-1,aproteinrecruitedbyserumresponsefactortothec-fosserumresponseelement.Cell68:597–612.

Durfee,T.,Becherer,K.,Chen,P.L.,Yeh,S.H.,Yang,Y.,Kilbburn,A.E.,Lee,W.H.&Elledge,S.J.(1993)Theretinoblastomaproteinassociateswiththeproteinphosphatasetype1catalyticsubunit.Genes Devel.7:555–569.

Estojak,J.,Brent,R.&Golemis,E.A.(1995)Correlationoftwo-hybridaffinitydatawithin vitromeasurements.Molecular and Cellular Biology15:5820–5829.

Feilotter,H.E.,Hannon,G.J.,Ruddel,C.J.&Beach,D.(1994)Constructionofanimprovedhoststrainfortwohybridscreening.Nucleic Acids Res.22:1502–1503.

Fields,S.(1993)Thetwo-hybridsystemtodetectprotein-proteininteractions.METHODS: A Companion to Meth. Enzymol.5:116–124.

Fields,S.&Song,O.(1989)Anovelgeneticsystemtodetectprotein-proteininteractions.Nature340:245–247.

Fields,S.&Sternglanz,R.(1994)Thetwo-hybridsystem:anassayforprotein-proteininteractions.Trends Genet.10:286–292.

Finley,Jr.,R.L.&Brent,R.(1994)InteractionmatingrevealsbinaryandternaryconnectionsbetweenDrosophila cellcycleregulators.Proc. Natl. Acad. Sci. USA 91:12980–12984.

Gietz,D.,St.Jean,A.,Woods,R.A.,&Schiestl,R.H.(1992)Improvedmethodforhighefficiencytransformationofintactyeastcells.Nucleic Acids Res.20:1425.

Golemis,E.A.,Gyuris,J.&Brent,R.(1996)Analysisofproteininteractions;andInteractiontrap/two-hybridsystemstoiden-tifyinteractingproteins.InCurrent Protocols in Molecular Biology(JohnWiley&Sons,Inc.),Sections20.0and20.1.

Guarente,L.(1993)Strategiesfortheidentificationofinteractingproteins.Proc. Natl. Acad. Sci. USA90:1639–1641.

Gubler,U.&Hoffman,B.J.(1983)AsimpleandveryefficientmethodforgeneratingcDNAlibraries.Gene25:263–269.

Guthrie,C.&Fink,G.R.(1991)Guidetoyeastgeneticsandmolecularbiology. InMethods in Enzymology (AcademicPress,SanDiego)194:1–932.

Gyuris,J.,Golemis,E.,Chertkov,H.&Brent,R.(1993)Cdi1,ahumanG1andSphaseproteinphosphatasethatassociateswithCdk2.Cell 75:791–803.

Hagen,F.S.,Gray,C.L.&Kuijper,J.L.(1988)AssayingthequalityofcDNAlibraries.BioTechniques6(4):340–345.

Harper,J.W.,Adami,G.R.,Wei,N.,Keyomarsi,K.&Elledge,S.J.(1993) Thep21Cdk-interactingproteinCip1isapotentinhibitorofG1cyclin-dependentkinases. Cell75:805–816.

Heslot,H.&Gaillardin,C.,eds.(1992)Molecular Biology and Genetic Engineering of Yeasts(CRCPress,Inc.).

Hill,J.,Donald,K.A.&Griffiths,D.E.(1991)DMSO-enhancedwholecellyeasttransformation.Nucleic Acids Res.19:5791.

Holm,C.(1993)Afunctionalapproachtoidentifyingyeasthomologsofgenesfromotherspecies.InMethods: A Com-panion to Methods in Enzymology5:102–109.

Holtz,A.E.&Zhu,L.(July1995)Clontechniques X(3):20.

Hopkin,K.(1996)Yeasttwo-hybridsystems:morethanbaitandfish.J. NIH Res.8:27–29.

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Ito,H.,Fukada,Y.,Murata,K.&Kimura,A.(1983)Transformationofintactyeastcellstreatedwithalkalications.J. Bacte-riol. 153:163–168.

Iwabuchi,K.,Li,B.,Bartel,P.&Fields,S.(1993)Useofthetwo-hybridsystemtoidentifythedomainofp53involvedinoligomerization.Oncogene8:1693–1696.

James,P.,Haliaday,J.&Craig,E.A.(1996)Genomiclibrariesandahoststraindesignedforhighlyefficienttwo-hybridselectioninyeast.Genetics144:1425–1436.

Kuo,H.J.,Maslen,C.L.,Keene,D.R.&Glanville,R.W.(1997)TypeVIcollagenanchorsendothelialbasementmembranesbyinteractingwithtypeIVcollagen. J. Biol. Chem.272:26522–26529.

Li,B.&Fields,S.(1993)Identificationofmutationsinp53thataffectitsbindingtoSV40Tantigenbyusingtheyeasttwo-hybridsystem.FASEB J.7:957–963.

Louret,O.F.,Doignon,F.&Crouzet,M.(1997)StableDNAbindingyeastvectorallowinghighbaitexpressionforuseinthetwo-hybridsystem.BioTechniques 23:816–819.

Luban,J.,Alin,K.B.,Bossolt,K.L.,Humaran,T.&Goff,S.P.(1992)Geneticassayformultimerizationofretroviralgagpolyproteins.J Virol.66:5157–5160.

Luban,J.&Goff,S.P.(1995)Theyeasttwo-hybridsystemforstudyingproteininteractions.Curr. Opinion. Biotechnol.6(1):59–64.

Ma,J.&Ptashne,M.(1987)Anewclassofyeasttranscriptionalactivators.Cell51:113–119.

Marcil,R.&Higgins,D.R.(1992)DirecttransferofplasmidDNAfromyeasttoE. coli byelectroporation.Nucleic Acids Res.20:917.

MatchmakerTwo-HybridSystem3(January1999)ClontechniquesXIV(1):12–14.

McNabb,D.S.&Guarente,L.(1996)Geneticandbiochemicalprobesforprotein-proteininteractions. Curr. Opin. Bio-technol.7(5):554–559.

Mendelsohn,A.R.&Brent,R.(1994)Biotechnologyapplicationsofinteractiontraps/two-hybridsystems.Curr. Opinion Biotechnol.5:482–486.

Moqadam,F.&Siebert,P.(1994)RapidAmplificationofthe3’endofcDNAswiththe3’-AmpliFINDERRACEKit.Clon-techniquesIX(3):6–9.

Ozenberger,B.A.&Young,K.H.(1995)Functionalinteractionofligandsandreceptorsofthehematopoieticsuperfamilyinyeast.Mol. Endocrinol.9:1321–1329.

Phizichy, E. M. & Fields, S. (1995) Protein-protein interactions: Methods for detection and analysis. Microbiol. Rev.59:94–123.

Ruden,D.M.(1992)Activatingregionsofyeasttranscriptionfactorsmusthavebothacidicandhydrophobicaminoacids.Chromosoma101:342–348.

Ruden,D.M.,Ma,J.,Li,Y.,Wood,K.&Ptashne,M.(1991)Generatingyeasttranscriptionalactivatorscontainingnoyeastproteinsequences.Nature 350:250–251.

Sambrook,J.,Fritsch,E.F.&Maniatis,T.(1989)Molecular Cloning: A Laboratory Manual(ColdSpringHarborLaboratory,ColdSpringHarbor,NY).

Scharf,S.J.(1990)CloningwithPCR.InPCR Protocols: A Guide to Methods and Applications.eds.Innis,M.A.,Gelfand,D.H.,Sninsky,J.J.&White,T.J.(AcademicPress,Inc.,SanDiego),pp.84–91.

Schiestl,R.H.&Gietz,R.D.(1989)Highefficiencytransformationofintactcellsusingsinglestrandednucleicacidsasacarrier. Curr. Genet.16:339–346.

Tirode,F.,Malaguti,C.,Romero,F.,Attar,R.,Camonis,J.&Egly,J.M.(1997)Aconditionallyexpressedthirdpartnerstabi-lizesorpreventstheformationofatranscriptionalactivatorinathree-hybridsystem.J. Biol. Chem.272:22995–22999.

vanAelst,L.,Barr,M.,Marcus,S.,Polverino,A.&Wigler,M.(1993)ComplexformationbetweenRASandRAFandotherproteinkinases.Proc. Natl. Acad. Sci. USA 90:6213–6217.

Vojtek,A.,Hollenberg,S.&Cooper,J.(1993)MammalianRasinteractsdirectlywiththeserine/threoninekinaseRaf. Cell74:205–214.

Ye,Q.,&Worman,H.J.(1995)Protein-proteininteractionsbetweenhumannuclearlaminsexpressedinyeast. Experi-mental Cell Res.219:292-298.

Yang,M.,Wu,Z.&Fields,S.(1995)Protein-peptideinteractionsanalyzedwiththeyeasttwo-hybridsystem.Nucleic Acid Res.23(7):1152–1156.

Zhang,X.,Settleman,J.,Kyriakis,J.M.,Takeuchi-Suzuki,E.,Elledge,S.J.,Marshall,M.S.,Bruder,J.T.,Rapp,U.R.&Avruch,J.(1993)Normalandoncogenicp21rasproteinsbindtotheamino-terminalregulatorydomainofc-Raf-1.Nature 364:308–313.

XII.Referencescontinued

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XIII.RelatedProducts

ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com

GAL4-basedOne-andTwo-HybridSystemsandRelatedProducts: • pGADT7ADVector 630442 • pGBKT7DNA-BDVector 630443 • MammalianMatchmaker™Two-HybridAssayKit 630301 • Matchmaker™PretransformedcDNALibraries many • Matchmaker™cDNAandGenomicLibraries many • Matchmaker™RandomPeptideLibrary 638853 • pBridgeVector 630404 • pCMV-Myc&pCMV-HAVectorSet 631604 • Matchmaker™Co-IPKit 630449 • Matchmaker™ADLD-InsertScreeningAmplimerSet 630433

Antibodies: • c-MycMonoclonalAntibody 631206 • c-MycMonoclonalAntibody-AgaroseBeads 631208 • HA-TagPolyclonalAntibody(AffinityPurified) 631207 • GAL4ADMonoclonalAntibody 630402 • GAL4DNA-BDMonoclonalAntibody 630403 GeneralReagentsforWorkingWithYeast: • Yeastmaker™YeastTransformationSystem2 630439 • Yeastmaker™CarrierDNA 630440 • Yeastmaker™YeastPlasmidIsolationKit 630441 • KC8ElectrocompetentandChemicallyCompetentCells 630435 630434 • YPDMedium 630409 • YPDAgarMedium 630410 • MinimalSDBase(containsglucoseorgalactose) 630411 630420 • MinimalSDAgarBase(containsglucoseorgalactose) 630412 630421 • DOSupplements many • X-α-Gal 630407

GeneralCloningReagents: • Advantage®2PolymeraseMix 639201 639202 • Advantage®2PCRKit 639206 639207 • NucleoSpin®ExtractionKit 635961



AppendixA.Media&SolutionRecipes

MediaforGrowthandSelectionofYeastClontechcarriesafulllineofyeastmediaincludingYPD,SDwithglucoseorgalactose;withorwithoutagar,andDropout(DO)SupplementsidealforusewithMatchmakerTwo-HybridSystemsandLibraries.PleaseseeSectionXIIIfororderinginformation.IfyoupurchasedyeastmediafromClontech,followthedirectionsprovidedwiththeproduct.Alternatively,youcanprepareyourownmediaandDOSupplementsusingthedetailedrecipesprovidedintheYPH,AppendixC.

• YPDAmedium

To1LofYPDMedium(Cat.No.630409),add15mloffilter-sterilized0.2%adeninehemis-ulfate(SigmaCat.No.A-9126)toafinalconcentrationof0.003%.

Reagentforlowstringencyscreens

• 65%glycerol/MgSO4solution(sterile)

FinalConc.

Glycerol 65%v/v

MgSO4 100mM

Tris-HCl(pH8.0) 25mM

MediaforTiteringandAmplifyingPlasmidLibrariesinE. coli • LBbroth

10g/L Bacto-tryptone

5g/L Bacto-yeastextract

5g/L NaCl

AdjustpHto7.0with5NNaOH.Autoclave.Storeatroomtemperature.

• LB/ampbroth

PrepareLBbroth,thenautoclaveandcoolto50°C.Addampicillinto100µg/ml.Storeat4°C.

• LB/ampplates

PrepareLBbroth, thenaddagar (18g/L),autoclave,andcool to50°C.Addampicillin to100µg/ml.Pourplatesandstoreat4°C.

• Ampicillinstocksolution(50mg/mlinH2O;1000X).Storeat–20°C.

X-α-Gal DissolveX-α-Galat20mg/mlindimethylformamide(DMF).StoreX-α-Galsolutionsinglass

orpolypropylenebottlesat–20°Cinthedark. 1.PouringX-α-Galindicatorplates a. Prepareandautoclave1.0Loftheappropriatedropoutagarmedium.Coolto55°C. b. Add1mlofX-α-Gal(20mg/ml). c.Pourplatesandallowmediumtohardenatroomtemperature. d. Platecellsandincubateattheappropriatetemperatureuntilbluecoloniesform. 2.SpreadingX-α-Galontopremadeplates a. DiluteX-α-Galto4mg/mlinDMF. b.Pourappropriatedropoutplatesandallowmediumtohardenat

roomtemperature.

c. Spread200µlofX-α-Gal(4mg/ml)ontoa15-cmplateor100µlontoa10-cmplateusingglassbeads.

Note:Toquickly(1–24hr)determineifayeaststraincontainsMEL1,spreadX-α-Galat20 mg/mlasdescribed.

d. Allowplatestodryfor15minatroomtemperature. e. Platecellsandincubateattheappropriatetemperatureuntilbluecoloniesform.



A. Important: • Dilutedlibrariesarealwayslessstablethanundilutedlibraries.Therefore,oncethelibrary

dilutionsaremade,usethemwithinthenexthourbeforedrasticreductionsintiteroccur. • Usepropersteriletechniquewhenaliquotingandhandlinglibraries. • Designanduseappropriatecontrolstotestforcross-contamination. • Always use the recommended concentration of antibiotic in the medium to ensure

plasmidstability. • pACTandpACT2librariesarereleasedfromλACTandλACT2libraries,respectively.Incubating

culturesofpACTandpACT2librariesat37°Ccanresultinplaquesonthehigh-densityplatesduetothepresenceofresidualphageinthelibrary.Theseplaquesshouldnotinterferewithlibrarytitering.However,iftheyarenumerous,retiterthelibraryat30–31°Candincubate36–48hr.

B. PlasmidLibraryTitering

ReagentsandMaterialsRequired:

• LBbroth(AppendixA) • LB/ampplates(100-mmplates;AppendixA) Note:Allowtheagarplatestodryunsleevedatroomtemperaturefor2–3days,orat30°Cfor3hr,priorto

platingcells.Moisturedropletsontheagarsurfacecanleadtounevenspreadingofcells.

• Sterileglassspreadingrod,bentPasteurpipette,or5-mmglassbeadsforspreadingcultureonplates.

1.Thawanaliquotofthelibrary,andplaceonice. 2.Mixbygentlevortexing.Transfer1µlto1mlofLBbrothina1.5-mlmicrocentrifugetube.

Mixbygentlevortexing.ThisisDilutionA(1:103). Note:pACTandpACT2librariesmaybeviscous,andrepeatedfreeze/thawcyclesmayincreaseviscosity.To

facilitateaccuratepipettingofthelibrary,firstdilutea10-µlsamplewith10µlofLBbroth.Thenpreparefurtherdilutionsfromthis1:1dilution.Besuretoaccountforthisextradilutionwhencalculatingthetiter.

3.Remove1µlfromDilutionA,andadditto1mlofLBbrothina1.5-mlmicrocentrifugetube.Mixbygentlevortexing.ThisisDilutionB(1:106).

4.Add1µlfromDilutionAto50µlofLBbrothina1.5-mlmicrocentrifugetube.Mixbygentlevortexing.SpreadtheentiremixtureontoaprewarmedLB/ampplate.

Note:Continuespreadingtheinoculumovertheagarsurfaceuntilallvisibleliquidhasbeenabsorbed.Thisprocedureisessentialforevengrowthofthecolonies.

5.Plate50-µland100-µlaliquotsofDilutionBonLB/ampplates. 6.Leaveplatesatroomtemperaturefor15–20mintoallowtheinoculumtosoakintotheagar. 7.Inverttheplates,andincubateat37°Cfor18–20hr,orat30–31°Cfor24–36hr. • ForpACTandpACT2libraries:incubateplatesat30–31°Cfor36–48hr. 8.Countthenumberofcoloniestodeterminethetiter(cfu/ml).Calculatethetiterasfollows: • DilutionA:No.coloniesx103x103=cfu/ml • DilutionB:(No.colonies/platingvolume)x103x103x103=cfu/ml Note:A2–5-foldrangeintitercalculationsisnotunusual,especiallyifmorethanonepersonisdoingthetitering.

AppendixB.LibraryTitering



YoumustamplifypremadeMatchmakerLibrariestoobtainenoughplasmidforlibraryscreeninginyeast.Youwillneed100–500µgofplasmidDNAtoscreen~1x106independentclones(seeTableIV).

Note:pACTandpACT2librarycustomers:Ifyouobservedplaquesonyourhigh-densitylibrarytiteringplates(AppendixB),youmaywishtousealowertemperature(i.e.,30–31°C)whenincubatingthelibraryamplificationplates.Thelowertemperaturewillrequirealongerincubationtime,asnotedbelow.IfyoufollowthisamplificationprotocolexactlythroughStep7,afewλplaquesontheamplificationplatesshouldnotaffectthequalityoryieldofplasmid.IfyouchoosetoincludeStep8,besuretoperformitat30°C.Lysisismorelikelytooccurinliquidcultures,andyourisklysingtheentirecultureattemperaturesover31.0°C.Growthat30–31°CisnotnecessaryoncethepACTorpACT2libraryDNAhasbeentransferredtoanewE. coli host.

A. ReagentsandMaterialsRequired • LB/ampagarplates(AppendixA)

Notes:• Theexactnumberofplatesrequireddependsonthesizeofthelibrary.Usethefollowingcalculationtodetermine

howmanyplatestouse.Normally,use3timesthesizeoftheoriginallylibraryandplateat20,000cfu/plate. (No.ofindependentclonesx3)=No.clonestoscreen 2x106x3=6x106clonestoscreen No.ofclonestoscreen/coloniesperplate=No.ofplates 6x106/20,000=300plates • Ifthetiteris6x108,determinetheamountofthelibrarystocktospreadoneachplate. No.clonestoscreen/librarytiter=μlsoflibrarytoplate 6x106/6x108titer=10μl • Calculatethevolumeofmedianeededtoplate150μloneachplate. 300platesx150μl=52.5ml • Add10μlofthelibraryto52.5mlofLBampandspread150 μlontoeachofthe300LBampplates • Allowtheagarplatestodryatroomtemperaturefor2–3days,orat30°Cfor3hr,priortoplatingcells.

Moisturedropletsontheagarsurfacecanleadtounevenspreadingofcells.

• LB/glycerol(1L;LBbrothcontaining25%glycerol) • Sterileglassspreadingrod,bentPasteurpipette,or5mmdiametersterileglassbeads(~10/

plate) • [Optional,forStep8]LB/ampbroth(2L;AppendixA)andsterile,50–80%glycerol.

B. PlasmidLibraryAmplificationProtocol 1.Ifyouhavenotdonesoalready,titertheplasmidlibrary(AppendixB). 2.PlatethelibrarydirectlyonLB/ampplatesatahighenoughdensitysothattheresulting

colonieswillbenearlyconfluent(~20,000–40,000cfuper150-mmplate).Plateenoughcfutoobtainatleast2–3Xthenumberofindependentclonesinthelibrary.

Notes:

• Thenumberofindependentclonesisthenumberofindependentcoloniespresentinthelibrarybeforeamplification.IfyouhavepurchasedalibraryfromClontech,thesizeofthelibraryisstatedonthePAC.

• Topromoteevengrowthofthecolonies,continuespreadingtheinoculumovertheagarsurfaceuntilallvisibleliquidhasbeenabsorbed,andthenallowplatestositatroomtemperaturefor15–20min.Ifusingglassbeadstospreadthecolonies,shaketheplatebackandforth—notroundandround.

3.Inverttheplates,andincubateat37°Cfor18–20hr. Notes:

• ForpACTorpACT2libraries:incubateplatesat30–31°Cfor36–48hr,oruntilconfluent. • Growingthetransformantsonsolidmediuminsteadofinliquidcultureminimizesunevenamplification

oftheindividualclones.

4.Add ~5 ml of LB/glycerol to each plate and scrape colonies into liquid. Pool all theresuspendedcoloniesinoneflaskandmixthoroughly.

Note:Toobtainhigheryields,scrapethecoloniesintoLB/amp(noglycerol)andpoolthecoloniesinoneflask.Incubateat30–31°Cfor2–4hrwithvigorousshaking(200rpm).Addsterileglycerolto25%,andproceedtoStep5.

AppendixC.PlasmidLibraryAmplification



5.Setasideone-thirdofthelibraryculture(roughlyequivalentto3Lofovernightculture)fortheplasmidpreparation;thisportioncanbestoredat4°Cifyouplantouseitwithin2weeks.Forstorage>2weeks,dividethecultureinto50-mlaliquots,andstoreat–70°C.

• Setasidefive1-mlaliquotsofthelibrarycultureincaseyouwishtore-amplifythelibraryatalatertime.Storethealiquotsat–70°C.

• Dividetheremainderofthelibrarycultureinto50-mlaliquotsandstoreat–70°C. 6.PrepareplasmidDNAusinganystandardmethodthatyieldsalargequantityofhighly

purifiedplasmid.(SeeSambrooketal.,1989forCsClgradientpurification,ifnecessary.)Note:ThecellculturefromStep4willbeverydense(OD600>>1),soadjusttheplasmidpreparationprotocolaccordingly(i.e.,followtheprocedureasifyouwereprocessing3Lofovernightliquidculture)orprepareasteriledilutionfrom10-1to10-3.PlasmidpreparationproceduresarebasedonacultureofOD600=1–2.Yourplasmid preparation must take into account the much higher than normal cellular concentration and bemagnifiedaccordingly.If30mlofculturewhendilutedto10-2hasanOD600=1,treatthestockasthoughitwere3LofcultureandprepareplasmidDNAusingaNucleoBondMegaorGigaPlasmidKit(Cat.Nos.635938&635939).

7.ExpectedyieldsofplasmidDNAper1x106cfu: • pACT&pACT2Libraries:0.25mg • allotherMatchmakerGAL4Libraries:~1mg 8.[Optional]Toobtainhigheryields,scrapethecolonies intoLB/amp(noglycerol)and

poolthecoloniesinoneflask.Incubateat31–30°Cfor2–4hrwithat200rpm.Addsterileglycerolto25%,thenproceedwithStep5above.

AppendixC.PlasmidLibraryAmplificationcontinued

Table of ContentsI. IntroductionII. Overview: A Yeast Two-Hybrid ScreenIII. Lists of ComponentsIV. Additional Materials RequiredV. Yeast Strains & PhenotypesA. Yeast Host StrainsB. Phenotypes

VI. Control VectorsA. Positive ControlsB. Negative Control

VII. Matchmaker GAL4 cDNA & Genomic LibrariesA. Library ConstructionB. Library Quality Control Information

VIII. Preparing for a Yeast Two-Hybrid ScreenA. Construct Fusion GenesB. Obtain or Construct an AD Fusion LibraryC. Verify that Constructs Do Not Activate Reporter GenesD. Verify Protein Expression

IX. Library Transformation & Screening ProtocolsA. Transformation ScalesB. Yeast Transformation ProtocolsC. Plating and Screening Transformation MixturesD. Calculations

X. Analysis & Verification of Putative Positive ClonesA. Retest PhenotypesB. Isolate Plasmid DNA from YeastC. Sort Colonies to Eliminate DuplicatesD. Rescue AD/Library Plasmids via Transformation in E. coliE. Retest Protein Interactions in YeastF. In vitro AnalysisG. Sequencing AD/Library InsertsH. In vivo AnalysisI. Additional Two-Hybrid Tests

XI. Troubleshooting GuideXII. ReferencesXIII. Related ProductsAppendix A. Media & Solution RecipesAppendix B. Library TiteringAppendix C. Plasmid Library AmplificationList of TablesTable I. List of AbbreviationsTable II. Matchmaker Yeast Strain GenotypesTable III. Matchmaker Yeast Strain PhenotypesTable IV. Matchmaker Two-Hybrid System 3 VectorsTable V. Comparison of Two-Hybrid Transformation MethodsTable VI. Setup for a Two-Hybrid Library Screen

List of FiguresFigure 1. Overview of Matchmaker System 3 Advances and ScreeningFigure 2. The Two-Hybrid PrincipleFigure 3. Reporter Constructs in Yeast Strains AH109 and Y187Figure 4. Overview of Performing a Yeast Two-Hybrid ScreenFigure 5. Screening an AD Fusion Library Using Strain AH109Figure 6. Strategies for Analyzing and Verifying Putative Positive ClonesFigure 7. Yeast mating to Verify Protein Interactions

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Matchmaker GAL4 Two-Hybrid System 3 & Libraries User … Manual/PT3247...2007/04/03 · MEL1 is an endogenous GAL4-responsive gene.The lacZ reporter gene was introduced into PJ69-2A