pET System Manual

1USAandCanadaUnitedKingdomandIrelandGermanyAllOtherCountriesTel8005267319UKFreephone0[e-mail][e-mail]@[e-mail]s6Growthmedia6Antibioticsandstocksolutions7C.VectorPreparation7D.InsertPreparation9III.CloningInsertsinpETVectors10A.Ligation10B.Transformation10Handlingtips11Procedure12Platingtechniques13ColiRollers™PlatingBeads14Standardspreader14C.StorageofStrains14D.AnalysisofpETRecombinants15Ligationscreening15Colonyscreening16Plasmidpreparationprocedure18IV.ExpressingtheTargetGene19A.BacteriophageCE619B.ExpressionHostTransformation19C.InductionofλDE3LysogenswithIPTG19Preparationforinduction19Sampleinductionprotocol20D.AutoinductionofλDE3Lysogens20OvernightExpress™InstantTBMedium21V.TargetProteinVerification24NormalizingloadingvolumesforSDS-PAGE24Opticaldensityanalysisoftheinducedculture24A.PopCulture®QuickScreenforExpressionLevel,Activity,andSolubility25B.TotalCellProtein(TCP)Fraction25C.MediumFraction25D.PeriplasmicFraction26E.SolubleCytoplasmicFraction28BugBuster®MasterMixtreatment28rLysozyme™Solutionandfreeze/thawtreatment29Mechanicaldisruption29F.InsolubleCytoplasmicFraction31InclusionbodypurificationafterBugBusterMasterMixtreatment31InclusionbodypurificationaftermechanicalcelllysisorrLysozymetreatment32G.PreparationofExtractswithPopCultureReagent32H.DetectingandQuantifyingTargetProteins34NormalizedSDS-PAGEgelloading35VI.PurifyingTargetProteins36A.ProteinPurification36B.SolubilizationandRefoldingProteins36VII.AdditionalGuidelines37A.ChoosingapETVector37Solubilityandcellularlocalization38Fusiontags39Antibioticresistance40B.pETVectorCloningStrategies43Producerecombinantproteinswithoutfusions43Producerecombinantproteinswithoutfusionsafterproteasecleavage44Ligation-independentcloning44C.RegulatingProteinExpressioninthepETSystem45TABLEOFCONTENTSNovagen•pETSystemManual•11thEditionTB05511thEdition01/062USAandCanadaUnitedKingdomandIrelandGermanyAllOtherCountriesTel8005267319UKFreephone0[e-mail][e-mail]@[e-mail]•pETSystemManual•11thEdition©2005EMDBiosciences,Inc.,anaffiliateofMerckKGaA,Darmstadt,Germany.TheNovagen®name,Novagenlogo,BugBuster®,His•Bind®,His•Tag®,HSV•Tag®,NovaTope®,PelletPaint®,PopCulture®,andT7•Tag®areregisteredtrademarksofEMDBiosciences,Inc.intheUnitedStatesandcertainotherjurisdictions.BetaRed™,Clonables™,ColiRollers™,DsbA•Tag™,DsbC•Tag™,Ekapture™,FRETWorks™,GST•Bind™,GST•Mag™,GST•Tag™,His•Mag™,LICDuet™,LumiBlot™,Lysonase™,Mobius™,NovaTaq™,Nus•Tag™,OnEx™,OvernightExpress™,PerfectDNA™,PerfectProtein™,pETBlue™,pETcoco™,RoboPop™,Rosetta™,RosettaBlue™,Rosetta-gami™,S•Tag™,Singles™,SpinPrep™,Strandase™,TrailMix™,TriEx™,Trx•Tag™,Tuner™,andXarrest™aretrademarksofEMDBiosciences,Inc.Fractogel®andBenzonase®areregisteredtrademarksofMerckKGaA,Darmstadt,Germany.MagPrepisaregisteredtrademarkofEMDChemical,Inc.Sonifier®isaregisteredtrademarkofBransonInstruments,Inc.MacroPrep®isaregisteredtrademarkofBio-RadLaboratories,GmbH,Ltd.Cy5™isatrademarkofGEHealth-care.Strep•Tactin®andStrep•Tag®areregisteredtrademarksofIBAGmbH.PicoGreen®isaregisteredtrademarkofMolecularProbes,Inc.Triton®isaregisteredtrademarkofRohmandHaasCo.Superflow™isatrademarkofSterogeneBioseparationsInc.SpeedVac®isaregisteredtrademarkofThermoSavant,Inc.BugStopper™isatrademarkofWhatmanInternational,Ltd.ThepETexpressionsystemiscoveredunderU.S.PatentNos.4,952,496;5,693,489;and5,869,320.Foracademicandnon-profitlaboratories,anondistributionagreementaccompaniestheproducts.Commerciallaboratoriesmustobtainaresearch-uselicensefromBrookhavenScienceAssociatespriortopurchaseoftheproducts.ThepET-32andpET-48vectorsaresoldunderpatentlicensefromGeneticsInstitute,Inc.Forresearchuseonly.LicensesforcommercialmanufactureorusemaybeobtaineddirectlyfromGeneticsInstituteInc.,87CambridgeParkDrive,Cambridge,MA02140.VectorscontainingtheHis•TagsequencearelicensedunderU.S.PatentNos.5,310,663;5,284,933;andEuropeanPatentNo.282,042issuedtoHoffmann-LaRoche,Inc.,NutleyNJand/orHoffmann,LaRoche,Ltd.Basel,Switerzerlandandareprovidedonlyforuseinresearch.InformationaboutlicenseforcommercialuseisavailablefromQIAGENGmbH,Max-Volmer-Str.4,D-40724Hilden,Germany.Ni-NTAHis•BindResinsaremanufac-turedbyQIAGENGmbH.His•TagMonoclonalAntibodyiscoveredunderGermanPatent,No.DE19507166andinternationalpatentapplicationsarefiledunderW09626963andisprovidedonlyforuseinresearch.InformationaboutlicenseforcommercialuseisavailablefromQIAGENGmbH,Max-Volumer-Str.4,D-40724Hilden,Germany.TheGST•TagtechnologyiscoveredunderU.S.PatentNo.5,654,176,EuropeanPatentNo.293,249B1,andAustralianpatentNo.607,511.Vect