首页 > Lucigen > Lucigen/OverExpress C43(DE3) Electrocompetent Cells/60345-1/12 rxns (SOLOs)

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Lucigen/OverExpress C43(DE3) Electrocompetent Cells/60345-1/12 rxns (SOLOs)

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ExpressgenesclonedintoanyT7vectorwiththeseBL21(DE3)derivativesEffectiveinexpressingtoxic&membraneproteinsCitedinover350researcharticlesInterestedinacompetentE.colistrainforroutineproteinexpression?LookhereE.coliBL21(DE3)strains,likeLucigen’sE.cloni®EXPRESSCompetentCellsprovidereliableexpressionofmanygenesclonedintoT7expressionvectors(e.g.,pETorLucigen’spSMART®-CDNAvectors).However,insomecasesexpressionisminimalornotdetectablebecausetherecombinantprotein,whenexpressed,isdeleteriousorlethaltothesestandardBL21strains.Examplesofsuchtoxicproteinsincludemanymembraneproteins,somecytoplasmicproteins,andnucleases.Unfortunately,successfulexpressionofoneormoretoxicproteinsisoftenimportanttotheexperimentalgoal.Lucigen’sOverExpressElectrocompetentandChemicallyCompetentCellsareE.colistrainsthatareeffectiveinexpressingtoxicproteinsfromallclassesoforganisms,includingeubacteria,yeasts,plants,viruses,andmammals.Theeffectivenessofthesenewstrainsinexpressingtoxicproteinshasbeenvalidatedinmorethan350publications.TheOverExpressstrainscontaingeneticmutationsphenotypicallyselectedforconferringtolerancetotoxicproteins.ThestrainC41(DE3)wasderivedfromBL21(DE3).Thisstrainhasatleastonemutation,whichpreventscelldeathassociatedwithexpressionofmanyrecombinanttoxicproteins.ThestrainC43(DE3)wasderivedfromC41(DE3)byselectingforresistancetoadifferenttoxicproteinandcanexpressadifferentsetoftoxicproteinstoC41(DE3).Figure1graphicallyillustratestheadvantagesoftheOverExpressCompetentCells,comparedtostandardBL21(DE3)cells,inexpressingtoxicproteins.Figure1.GreenFluorescentProtein(top)orRedFluorescentProtein(bottom)expressedfromaT7promoterconstructthatwastransformedintoC41,BL21,orC43competentcellsspreadonIPTGplatestoinduceproteinexpression.Table1andFigure2summarizetransformationeffectiveness,toleranceofexpression-inducedtoxicity,andproteinexpressionforT7expressionplasmidscodingforavarietyofrecombinantproteins.TheseresultsdemonstratethattheOverExpressC41(DE3)andC43(DE3)strainsareclearlysuperiortotheparentalBL21(DE3)intransformationandexpressionoftoxicproteins.Table1.ComparisonofOverExpressC41(DE3)andC43(DE3)cellswiththeparentalstrainBL21(DE3)intransformationandexpressionofheterologousproteins.**StrainTransformationSuccessRateaExpression-inducedToxicitybExpressingPlasmidscBL21(DE3)16/26(62%)25/26(96%)14/26(54%)C41(DE3)28/28(100%)14/28(50%)24/28(86%)C43(DE3)28/28(100%)1/28(4%)23/28(81%)Figure2.ComparisonofOverExpressC41(DE3)andC43(DE3)cellswiththeparentalstrainBL21(DE3)intransformationandexpressionofheterologousproteins.**aTransformationsuccesscorrespondstothepresenceofcoloniesonLB+ampicillinagarfollowingtransformationwithaplasmid.bExpressiontoxicitycorrespondstotheabsenceofcoloniesonLB+ampicillin+IPTGagarfollowingtransformationwithaplasmid.cExpressingplasmidscorrespondstoobservationofaheterologousproteininthetotalcellpelletonCoomassie-stainedSDS-PAGEfollowinggrowthofacolonyinLB+ampicillinmediumandinductionwithIPTG.**L.Dumon-Seignovert,G.Cariot,andL.Vuillard(2004).ProteinExpressionandPurification37,203-206.Datausedwithpermission.AsinstandardBL21(DE3)strains,OverExpressC41(DE3),C41(DE3)pLysS,C43(DE3),andC43(DE3)pLysSarelysogensof&lamBDa;DE3.ThesestrainscarryachromosomalcopyoftheT7RNAPolymerasegeneunderthecontrolofthelacUV5promoter.ThesestrainsaresuitableforproductionofproteinfromtargetgenesclonedintoT7-drivenexpressionvectors.OverExpressC41(DE3),C41(DE3)pLysS,C43(DE3),andC43(DE3)pLysSarealsodeficientinthelonandompTproteases.OverExpressC41(DE3)pLysSandC43(DE3)pLysSalsocarryachloramphenicol-resistantplasmidthatencodesT7lysozyme,whichisanaturalinhibitorofT7RNApolymerase.CellscontainingpLysSproduceasmallamountofT7lysozyme.ThesestrainsareusedtosuppressbasalexpressionofT7RNApolymerasepriortoinduction,thusstABIlizingrecombinantsencodingparticularlytoxicproteins.FAQWhichOverExpresscellstrainshouldIuse?ItisdifficulttopredictwhichofthefourOverExpressstrains–C41(DE3),C43(DE3),C41(DE3)pLysS,orC43(DE3)pLysS–willworkbestinexpressingagivenprotein.WerecommendinitiallyusingtheOverExpressComboPack™whichcontains3reactionseachofthefourOverExpresscompetentcellstrains,todeterminewhichoneisbestforyourapplication.TheOverExpressstrainsareavailableaselectrocompetentorchemicallycompetentcells.Becausetherearenointrinsicantibioticresistances(orplasmids)ineitherC41(DE3)orC43(DE3),thestrainscanbedifferentiatedfromeachotherandfromBL21(DE3)bytransformationwithastrainverificationvector,pAVD10.pAVD10containstheuncFgene(encodingthebeta-subunitofE.coliATPase)underthecontroloftheT7promoter.ThisplasmidislethaltoBL21(DE3)andtoinducedC41(DE3),butitistoleratedbyC43(DE3)regardlessofinduction.pAVD10isprovidedwithOverExpressCells.ORDERINFORMATIONEachOverExpresskitcontainsElectrocompetentorChemicallyCompetentCellsinSOLOpackaging(1transformationpertube),ExpressionRecoveryMedium(lactoseminus),pUC19PositiveControlPlasmid,pAVD10VerificationPlasmid,andcompleteprotocols.ComboPackscontain3reactionseachofchemicallycompetentC41(DE3),C43(DE3),C41(DE)pLysS,andC43(DE3)pLysS.

美国Lucigen公司，自1998年成立至今，一直致力于生命科学领域相关科研产品的研究与开发，在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品，包括：CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术，过硬的产品质量，良好的产品服务赢得了全球广大用户的信赖。