pET表达系统说明书(Novagen公司)

pETSystemManualTB0559thEdition05/00Novagen1UnitedStates&Canada800-207-0144Germany08006931000UnitedKingdom0800622935OryourlocalsalesofficeTableofContentsTableofContents1I.AbouttheSystem3A.Description3B.LicensingandUseAgreement3C.SystemComponents3D.ThepETVectors4VectorCharacteristicsandCloningStrategy4Ligation-IndependentCloning(LIC)ofPCRProducts4FusionTags5E.AntibioticResistance6F.pETVectorCharacteristics7G.HostsforCloning8H.HostsforExpression8pETSystemHostStrainCharacteristics9I.SelectingHostStrains11ListofpETSystemHostStrainsandLambdaPhages12J.MediaContainingGlucose14K.TheT7lacPromoter14L.pLysSandpLysEHosts14M.pLacIHosts15N.BacteriophageCE615O.InductionControls16II.GettingStarted17A.ThepETSystemProcess17B.GrowthMedia18C.StorageofStrains19D.VectorPreparation20Recommendations20E.InsertPreparation21III.CloningInsertsinpETVectors22A.Ligation22B.Transformation22HandlingTips23Procedure23PlatingTechnique24C.AnalysisofpETRecombinants24Transcription/TranslationAnalysiswithEcoPro™orSTP3®Systems25PlasmidTemplates25PCRTemplates25LigationPCRforTranscription/TranslationAnalysis26ColonyPCRforTranscription/TranslationAnalysis26ColonyScreening27PlasmidMiniprepProcedure28Sequencing29IV.ExpressingtheTargetGene30A.ExpressionHostTransformation30B.InductionofλλλλDE3Lysogens30PreparationforInduction30SampleInductionProtocol30C.OptimizingExpression31PlasmidStabilityTest31D.Solubility31FormationofDisulfideBonds32E.ToxicGenesandPlasmidInstability32pETSystemManual2NovagenTB0559thEdition05/00UnitedStates&Canada800-207-0144Germany08006931000UnitedKingdom0800622935OryourlocalsalesofficeUseofAmpicillin33PrecautionstoMaximizeExpression33RationaleforPlasmidStabilityTest34F.DifficultTargetProteins34OtherFactorsInfluencingExpressionLevel36V.DetectingandQuantifyingTargetProteins37Detection/AssayProductsforFusionTags37VI.PurifyingTargetProteins38A.SmallScaleAnalysis38GrowthandInduction38OpticalDensityAnalysisoftheInducedCulture39TotalCellProtein(TCP)Sample39MediaSample40PeriplasmicFractionSample–OsmoticShock40SolubleCytoplasmicFraction41InsolubleCytoplasmicFraction42B.PreparationofExtractswithBugBuster™ReagentandBenzonase®Nuclease42BugBuster/BenzonaseSolubleFraction42BugBuster/BenzonaseInclusionBodyPurification43C.SDS-PAGEandWesternBlotAnalysis45NormalizedSDS-PAGEGelLoading45D.LargeScaleInductionandFractionation46MediaFraction46PeriplasmicFraction46SolubleWholeCellExtractFraction46InsolubleWholeCellExtractFraction–IsolationofInclusionBodies47SolubilizationofInclusionBodiesandRefoldingProteins48VII.InductionControl:ββββ-GalactosidaseRecombinant49ββββ-GalactosidaseAssay49VIII.Acknowledgments50IX.References50X.Index52XI.AcademicandNon-profitLaboratoryAssuranceLetter55XII.AD494,BL21trxB,OrigamiandOrigamiBNon-DistributionAgreement55XIII.RelatedProductsandSeparateComponents56Copyright
1992–2000byNovagen,Inc.Allrightsreserved.BugBuster,CBD•Tag,Clonables,Dsb•Tag,EcoPro,EKapture,EXlox,FRETWorks,GST•Bind,GST•Tag,His•Bind,His•Tag,HSV•Tag,LumiBlot,Mobius,NovaTope,NusTag,Origami,PelletPaint,PerfectDNA,PerfectProtein,pETBlue,pSCREEN,S•Tag,SingleTubeProtein,Singles,SpinPrep,STP3,Strandase,T7•Tag,TriEx,Trx•Tag,Tuner,UltraMobius,Xarrest,theNovagennameandlogoaretrademarksandregisteredtrademarksofNovagen,Inc.BenzonaseisatrademarkofBenzonPharmaA/S.SuperflowisatrademarkofSterogeneBioseparationsInc.FractogelisatrademarkofMerckKGaA,Darmstadt,Germany.MagPrepisatrademarkofEMIndustriesInc.CBINDisatrademarkofCBDTechnologies,Inc.Novagen’sprimersaredesignedandsoldforuseinthePolymeraseChainReaction(PCR)processcoveredbypatentsownedbyHoffmann-LaRoche.UseofthePCRprocessrequiresalicense.AlicenseforresearchmaybeobtainedbypurchaseanduseofauthorizedreagentsandDNAthermalcyclersfromthePerkin-ElmerCorporationorbyotherwisenegotiatingalicensewithPerkin-Elmer.TritonisatrademarkofRohmandHaasCo.ThepETsystemiscoveredbyU.S.Patentno.4,952,496.Anon-distributionagreementaccompaniestheproducts.CommercialcustomersmustobtainalicenseagreementfromBrookhavenScienceAssociatesbeforepurchase.ThepET-32vectorsaresoldunderpatentlicensefromGeneticsInstitute,Inc.Forresearchuseonly.LicensesforcommercialmanufactureorusemaybeobtaineddirectlyfromGeneticsInstitute,Inc.,87CambridgeParkDrive,Cambridge,MA02140.TheCBD•TagtechnologyiscoveredunderU.S.Patentnos.5,496,934;5,202,247;5,340,731;and5,137,819.UseofthistechnologyforcommercialpurposesrequiresalicensefromCBDTechnologies,Inc.Ni-NTAHisBindResinsaremanufacturedbyQiagenGmbH.HisTagMonoclonalAntibodyiscoveredunderGermanPatentNo.DE19507166.TheGST•TagtechnologyiscoveredunderU.S.Patentno.5,654,176,EuropeanPatentno.293,249B1,andAustralianPatentno.607,511.VectorscontainingtheHisTagsequencearelicensedunderU.S.PatentNos.5,310,663;5,284,933;andEuropeanPatentNo.282,042issuedtoHoffmann-LaRoche,Inc.,NutleyNJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandareprovidedonlyforuseinresearch.InformationaboutlicensesforcommercialuseisavailablefromQIAGENGmbH,Max-Volmer-Str.4,D–40724Hilden,Germany.pETSystemManualTB0559thEdition05/00Novagen3UnitedStates&Canada800-207-0144Germany0800