噬菌体展示肽库说明书

InstructionManualPh.D.™PhageDisplayLibrariesProteIntooLSneB#e8100S,#e8101S,#e8102L,#e8110S,#e8111L,#e8120S,#e8121LStoreat–20°C1tableofContents:Introduction..........................................................................2MediaandSolutions................................................................6GeneralM13Methods..............................................................7ConstructionofpIII-DisplayLibrariesusingM13KE............................9PanningProtocolsProtocol1:SurfacePanningProcedure(DirectTargetCoating).........14Protocol2:Solution-phasePanningwithaBiotinylatedTargetandStreptavidinPlateCapture...............................................18Protocol3:Solution-phasePanningwithAffinityBeadCapture.........19PostPanningProtocolsPlaqueAmplificationforELISAorSequencing............................22SequencingofPhageDNARapidPurificationofSequencingTemplates............................23PhageELISABindingAssaywithDirectTargetCoating...................25UseofSyntheticPeptidesinSpecificityAnalysis..........................27ExpressionofSelectedSequencesasMonovalentMBPFusions................27Appendix:OptimizingPeptideBindingInteractions....................................29ChoiceofLibrary..............................................................30SelectionofCell-SpecificPeptides..........................................31Troubleshooting...................................................................32References.........................................................................38OrderingInformation..............................................................41Ph.D.™PhageDisplayLibraries2KitComponentsAllkitcomponentsshouldbestoredat–20°Cexceptwherenoted:PhageDisplayPeptideLibrary100µl,~1x1013pfu/ml.SuppliedinTBSwith50%glycerol.-96gIIIsequencingprimer5´-HOCCCTCATAGTTAGCGTAACG–3´,100pmol,1pmol/µl-28gIIIsequencingprimer5´-HOGTATGGGATTTTGCTAAACAAC–3´,100pmol,1pmol/µlE.coliER2738hoststrainF´proA+B+lacIqΔ(lacZ)M15zzf::Tn10(TetR)/fhuA2glnVthiΔ(lac-proAB)Δ(hsdMS-mcrB)5(rk–mk–McrBC–).Hoststrainsuppliedas50%glycerolcul-ture;notcompetent.Storeat–70°C.Streptavidin,lyophilized1.5mgBiotin,10mM100µlPh.D.PeptideDisplayCloningSystem(SuppliedwithE8101only)20µgM13KEglllCloningVector,2150pmolExtensionPrimerIntroductionPhagedisplaydescribesaselectiontechniqueinwhichalibraryofpeptideorproteinvariantsisexpressedontheoutsideofaphagevirion,whilethegeneticmaterialencodingeachvariantresidesontheinside(1-3).ThiscreatesaphysicallinkagebetweeneachvariantproteinsequenceandtheDNAencod-ingit,whichallowsrapidpartitioningbasedonbindingaffinitytoagiventargetmolecule(antibodies,enzymes,cell-surfacereceptors,etc.)byaninvitroselectionprocesscalledpanning(4).Initssimplestform,panningiscarriedoutbyincubatingalibraryofphage-displayedpeptidesonaplate(orbead)coatedwiththetarget,washingawaytheunboundphage,andelutingthespecificallyboundphage(Figure1).Theelutedphagearethenamplifiedandtakenthroughadditionalbinding/amplificationcyclestoenrichthepoolinfavorofbindingse-quences.After3–4rounds,individualclonesarecharacterizedbyDNAsequenc-ingandELISA.ThePh.D.™(phagedisplay)systemisbasedonasimpleM13phagevector,modifiedforpentavalentdisplayofpeptidesasN-terminalfusionstotheminorcoatproteinpIII(5-7).ThisproteinmodulatesphageinfectivitybybindingtotheF-pilusoftherecipientbacterialcell,andispresentin5copiesclusteredatoneendofthematureM13virion(2,8).Ifthedisplayedpeptideissufficientlyshort(50residues),theinfectivityfunctionofpIIIisnotaffected,andall5copiescancarrydisplayedpeptideswithoutmeasurableattenuationofphageinfectiv-ity(6).Asaresult,theM13KEgenomecontainsonlyasinglecopyofgeneIII3(gIII).Thisisincontrasttophagemidsystems,whichprovidebothfusedandunfusedcopies.ThereducedvalencyofpIIIlibrariescomparedtopVIIIlibrar-iesrendersthePh.D.systemmoresuitableforthediscoveryofhigheraffinityligands(Kdof10µMorbetter).ThecloningvectorM13KEisderivedfromM13mp19,allowingconstructionandrapidpropagationofphagedisplaylibrariesusingstandardM13tech-niques,withouttheneedforantibioticselectionorhelperphagesuperinfection(7).Extensivesequencingofnaïvelibrariespreparedinthisvectorsystemhasrevealedlittlesequencebiasapartfromselectionagainstunpairedcysteineresidues(unpublishedobservations)andtheexpectedreducedlevelsofarginine(butnotlysine)residues.ThereducedargininelevelsarelikelycausedbythesecY-dependentsecretionofpIII,andcanbeovercomeifdesiredbytheuseofaprlAsuppressorstrainforlibraryamplification(10).NewEnglandBiolabscurrentlyoffers3pre-maderandompeptidelibraries,aswellasthecloningvectorM13KEforconstructionofcustomlibraries.Figure1:Alibraryofphage,eachdisplayingadifferentpep-tidesequence,isexposedtoaplatecoatedwiththetarget.Unboundphagearewashedaway.Specifically-boundphageareelutedwithanexcessofaknownligandforthetarget,orbyloweringpH.After3rounds,individualclonesareisolatedandsequenced.+PanningwithapentavalentpeptidelibrarydisplayedonpIII.4Thepremadelibraries(Figure2)consistoflinearheptapeptide(Ph.D.-7)anddodecapeptide(Ph.D.-12)libraries,aswellasaloop-constraine