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公司介绍
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
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Lucigen/OverExpress C43(DE3) Chemically Competent Cells/60446-2/24 rxns (SOLOs)

Lucigen/OverExpress C43(DE3) Chemically Competent Cells/60446-2/24 rxns (SOLOs)
  • Lucigen/OverExpress C43(DE3) Chemically Competent Cells/60446-2/24 rxns (SOLOs)
商品介绍
  • 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.

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%)

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.
品牌介绍

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。

Lucigen Corporation致力于通过为生命科学和医疗保健专业人员提供优质的产品和服务来改善人们的生活。Lucigen于1998年在威斯康星州成立,提供的产品和服务使生命科学专业人士能够更有效地进行研究和测试。该公司已发展为向全球客户制造和销售超过325种生物医学研究产品和服务,所有这些产品和服务均遵循符合ISO 13485的质量体系核心能力包括酶进化,蛋白质表达,克隆,感受态细胞,下一代测序和分子诊断。

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