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儀器網(wǎng)> 資料庫(kù)>Invitrogen pGene v5-His 說明書

Invitrogen pGene v5-His 說明書

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2018-09-29 10:03 1678閱讀次數(shù)

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GeneSwith哺乳動(dòng)物誘導(dǎo)系統(tǒng)InvitrogenpGenev5-His產(chǎn)品參數(shù):MammalianSelection:ZeocinConstitutive/Inducible:InducibleExpressionLevelTightlycontrolled(activatewithmifepristone)SequencingPrimerpGeneFwdSequencingPrimerSequence:5'd[CTGCTATTCTGCTCAACCT]3'ProteinTags:6XHis,V5載體抗性:氨芐青霉素(Ampicillin)載體描述: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

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