Invitrogen pGene v5-His 说明书

文档简介GeneSwith哺乳动物诱导系统InvitrogenpGenev5-His产品参数:MammalianSelection：ZeocinConstitutive/Inducible：InducibleExpressionLevelTightlycontrolled（activatewithmifepristone）SequencingPrimerpGeneFwdSequencingPrimerSequence：5'd[CTGCTATTCTGCTCAACCT]3'ProteinTags：6XHis，V5载体抗性:氨苄青霉素（Ampicillin）载体描述:Switch-OnExpressionfromtheLowestBasalLevelsTheGeneSwitchSystemforinduciblemammalianexpressionisidealforexperimentsthatrequiretheabsolutelowestuninducedexpressionlevels.TheexpressionvectorpGene/V5-Hisprovidesaminimalpromoter,GAL4-E1b,consistingofthebindingsitesfortheyeastGal4DNAbindingproteinfollowedbytheTATAsequencefromtheAdenovirusE1bpromoter.Withoutadditionalfactors,theGAL4-E1bpromoteristranscriptionallysilent.ToactivatetranscriptionfromtheGAL4-E1bpromoter,theGeneSwitchregulatoryproteinisexpressedfromaminimalTKpromoteronthepSwitchvector.TheGeneSwitchproteinhasthreefunctionaldomains:Gal4DNABindingDomain（Gal4-DBD）tobindtheregulatoryproteintotheGAL4-E1bpromoterTruncatedhumanProgesteroneReceptorLigandBindingDomain（hPR-LBD）thatundergoesaconformationalchangewhenitbindstheprogesteroneantagonist,mifepristoneTranscriptionactivationdomainfromtheNFBtranscriptionfactorp65（p6D）toactivatetranscriptionfromthesilentGAL4-E1bpromoterIntheabsenceofmifepristone,theconformationofthehPR-LBDregionpreventstheGeneSwitchregulatoryproteinfromactivatingtranscriptionfromtheGAL4-E1bpromoter.WhenmifepristoneisaddedandbindsthehPR-LBDregion,theGeneSwitchregulatoryproteinassumesaconformationthatpermitsittostimulatetranscriptionfromtheGAL4-E1bpromoter.InductionoftheGeneSwitchSystemleadstoactivationofthegeneofinterestonthevectorpGene/V5-His.Inaddition,fourGal4bindingsitesupstreamoftheminimalHSVTKpromoteronpSwitchcanbindthepSwitchregulatoryprotein.Therefore,addingmifepristoneup-regulatesproductionoftheregulatoryprotein.TheincreasedlevelsoftheGeneSwitchregulatoryproteinresultininductionofthegeneofinterestfrompGene/V5-Histolevelsthatcanapproachthoseofviralpromoters.TheGeneSwitchKitoffersexceptionallylowuninducedandhighinducedexpressionlevelsinmammaliancells.ThesystemprovidestheexpressionvectorpGene/V5-Hiswiththeminimal,syntheticGAL4-E1bpromoterthatistranscriptionallysilentuntilactivated.TheGeneSwitchregulatoryproteinbindstheGAL4-E1bpromotertoactivatetranscriptionupontheadditionofmifepristone.Inaddition,theGeneSwitchproteinupregulatesitsownexpression,leadingtoamplifiedexpressionofthegeneofinterestfrompGene/V5-His.pGene/V5-Hisoffersseveralfeaturesthatfacilitateexpressionanalysisandpurificationofrecombinantproteinsinmammaliancells:C-terminalV5epitopetagfordetectionwithAnti-VntibodiesC-terminalpolyhistidine（6xHis）tagforrapidpurificationwithnickel-chelatingresinanddetectionwithAnti-His（C-term）AntibodiesTheZeocinresistancegeneforeffectiveselectioninbothmammaliancellsandE.coli