E.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.

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Table1andFigure2summarizetransformationeffectiveness,toleranceofexpression-inducedtoxicity,andproteinexpressionforT7expressionplasmidscodingforavarietyofrecombinantproteins.TheseresultsdemonstratethattheOverExpressC41(DE3)andC43(DE3)strainsareclearlysuperiortotheparentalBL21(DE3)intransformationandexpressionoftoxicproteins.

Table1.ComparisonofOverExpressC41(DE3)andC43(DE3)cellswiththeparentalstrainBL21(DE3)intransformationandexpressionofheterologousproteins.**

Strain	Transformation SuccessRatea	Expression-inducedToxicityb	ExpressingPlasmidsc
BL21(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%)

^aTransformationsuccesscorrespondstothepresenceofcoloniesonLB+ampicillinagarfollowingtransformationwithaplasmid.
^bExpressiontoxicitycorrespondstotheabsenceofcoloniesonLB+ampicillin+IPTGagarfollowingtransformationwithaplasmid.
^cExpressingplasmidscorrespondstoobservationofaheterologousproteininthetotalcellpelletonCoomassie-stainedSDS-PAGEfollowinggrowthofacolonyinLB+ampicillinmediumandinductionwithIPTG.
**L.Dumon-Seignovert,G.Cariot,andL.Vuillard(2004).ProteinExpressionandPurification37,203-206.Datausedwithpermission.

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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.

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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.

ORDERINFORMATION

EachOverExpresskitcontainsElectrocompetentorChemicallyCompetentCellsinSOLOpackaging(1transformationpertube),ExpressionRecoveryMedium(lactoseminus),pUC19PositiveControlPlasmid,pAVD10VerificationPlasmid,andcompleteprotocols.ComboPackscontain3reactionseachofchemicallycompetentC41(DE3),C43(DE3),C41(DE)pLysS,andC43(DE3)pLysS. Lucigen基因组编辑和工程面临许多挑战。为您的编辑实验获取可靠的试剂不应该是其中之一。无论您是在突破CRISPR技术的界限，在体内产生基因敲除或敲入还是在进行序列消化或鉴定的体外反应，您都需要可靠的工具和酶。依靠CRISPRcraft™获得质量，可靠性和稳定的性能。