- ExpressgenesclonedintoanyT7vectorwiththeseBL21(DE3)derivatives
- Effectiveinexpressingtoxic&membraneproteins
- Citedinover350researcharticles
- InterestedinacompetentE.colistrainforroutineproteinexpression?Lookhere
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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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.**
| 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%) |
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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.
ORDERINFORMATION
EachOverExpresskitcontainsElectrocompetentorChemicallyCompetentCellsinSOLOpackaging(1transformationpertube),ExpressionRecoveryMedium(lactoseminus),pUC19PositiveControlPlasmid,pAVD10VerificationPlasmid,andcompleteprotocols.ComboPackscontain3reactionseachofchemicallycompetentC41(DE3),C43(DE3),C41(DE)pLysS,andC43(DE3)pLysS.Lucigen
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Lucigen Corporation is working to improve people’s lives by providing quality products and services to life science and healthcare professionals. Founded in Wisconsin in 1998, Lucigen offers products and services that enable life-science professionals to perform their research and testing more efficiently and effectively. The company has grown to manufacture and sell over 325 biomedical research products and services to customers worldwide, all under an ISO 13485 compliant quality system. Core competencies include enzyme evolution, protein expression, cloning, competent cells, next gen sequencing, and molecular diagnostics.
产品列表：
No. | 品牌 | 货号 | 名称 | 规格 |
1 | lucigen | 40041-2 | CloneSmart HCAmp Blunt Cloning Kit | 20 rxns |
2 | lucigen | 40300-2 | CloneSmart LCAmp Blunt Cloning Kit | 20 rxns |
3 | lucigen | RG90910K | RiboGuard™ RNase Inhibitor | 10,000 Units |
4 | lucigen | AS3107 | AmpliScribe™ T7 High Yield Transcription Kit | 50 Reactions |
5 | lucigen | ASB71110 | AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit | 10 Reactions |
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