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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/Expresso® T7 SUMO Cloning and Expression System/49003-2/10 rxns

Lucigen/Expresso® T7 SUMO Cloning and Expression System/49003-2/10 rxns
  • Lucigen/Expresso® T7 SUMO Cloning and Expression System/49003-2/10 rxns
商品介绍

Solubleproteinhasneverbeenthisfastandeasy!

  • Enzyme-freedirectionalPCRcloninginseconds!
  • Savedaysofeffortwithready-to-usevectorsandcompetentcells-NOligationstep.
  • Tightly-controlledexpressionofN-terminal6xHis-taggedproteinswithcleavableSUMOsolubilitytag.
  • SystemsavailablewithexpressionunderthecontrolofT7ortunableRhamnosepromoters.

CompleteCloningandProteinExpressionSystem

TheExpressoSUMOCloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandsolubleexpressionofPCR-amplifiedgenes.Forproteinsthatforminclusionbodiesoraredifficulttoexpressinsolubleform,thepETite®N-HisSUMOandpRham™N-HisSUMOVectorsallowexpressionoftargetproteinswithanamino-terminal6xHis-SUMOproteintag.SUMO(smallubiquitin-likemodifier)isarelativelysmall(100-residue)polypeptidethathasbeenshowntoenhancethesolubleexpressionofmanyproteinsthatareotherwisedifficulttoproduceinE.coli.

Comparison Expresso and TraditionalTheExpressoSUMOCloningandProteinExpressionSystemsarebasedontheoriginalExpressoT7andExpressoRhamnoseCloningandProteinExpressionSystems,whichuseExpressioneeringTechnology™toprovideeffortlesshigh-efficiencycloningandtightlycontrolledproteinexpression.TheT7Systemiscompletewithpre-processedpETiteN-HisSUMOcloningvector,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidproteinexpressionproblemsseenwithleakyT7promoter-basedexpressionplasmidsystems.TheRhamnoseSystemiscompletewithpre-processedpRhamN-HisSUMOcloningvectorandhigh-efficiencyE.cloni10GCompetentCells,whichareusedforcloningandexpression,enablinghigherproteinexpressionthroughput.TheN-terminal6xHisSUMOtaggedrecombinantproteinscanberapidlypurifiedusingstandardNickelchromatography.SUMOExpressProteaseisincludedtoprovideefficientcleavageoftheSUMOtag,preciselyatthejunctionbetweentheSUMOtagandthetargetprotein.

Five-SecondDirectionalCloningofPCR-AmplifiedGenes

TheExpressoSUMOCloningandProteinExpressionSystemsuseExpressioneeringTechnology,theenzyme-freerecombinationalcloningstrategyoftheExpressosystemtoseamlesslyintegratethegenewiththeexpressionplasmid.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETiteorpRhamN-HisSUMOexpressionplasmids,andtransformimmediatelyintotheChemicallyCompetentCellsprovided(Figure1). 

 

SUMOVectorsincludes:

  1. StrongT7promoterforhigh-level,orRhamnosepromoterfortunable,recombinantproteinexpression.

  2. IncreasedsolubilityandfastproteinpurificationfromN-terminal6xHisSUMOfusiontag

  3. Smallsize(2.5kb)foreasierdownstreammanipulation.

  4. PatentedCloneSmart®technologyincreasescloningefficiency.

     

SUMO Vector Map

Figure2.SUMOexpressionvector:Smallsize(2.5kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Expressionplasmidispre-processedforinstantenzyme-freecloningofPCRproducts.

RescueinclusionbodiesandinsolubleproteinwithSUMOproteintag:

WehaveusedtheExpressoT7andExpressoT7SUMOCloningandExpressionSystemsforexpressionandpurificationofavarietyofproteins.Someresultsofanongoinglarge-scaleexpressionstudytoidentifyhydrolaseenzymesfromFibrobactersuccinogenesarepresentedinFigure3.Initially,48geneswereselectedforexpressiontrialsandclonedintothepETiteT7C-HisVector.Approximatelyhalfofthesecloneshaveproducedsoluble,activehydrolaseprotein,whileinotherinstancestargetproteinswereexpressedinaninsolubleform.FiveofthegenesproducinginsolubleproteinswerereamplifiedandclonedintothepETiteSUMOvector.WhentheresultingcloneswereexpressedinHI-ControlBL21(DE3)cells,recoveryofactiveproteininthesolublefractionwassignificantlyimprovedinfourofthefivecases(Table1).Althoughtagremovalwasnotnecessaryforhydrolaseactivity,thetagcouldberemovedefficientlybySUMOExpressProtease.

 

Expresso SUMO

Figure3.Large-scalecloningandexpressioncasestudy:(A)PCRproductsfrom48putativehydrolasegenesrangingfrom~1to>3kb.(B)Uninduced(-)andIPTG-induced(+)samplesofHI-ControlBL21(DE3)cellswith6differentgenesclonedintothepETiteC-HisVector.(C)EnhancedsolubilityofSUMO-tagged2201and2442geneproducts.Totalcellextractandsolublefractionsareshown.(D)Removalof6xHis-SUMOtagfrompurifiedSUMO-2201fusionproteinbySUMOprotease.–prot:uncleavedSUMO-2201fusionproteinafterIMACpurification;+prot:SUMOprotease-treatedfusionprotein;C:isolated2201proteinafterremovalof6xHis-SUMOfragmentandSUMOproteasebysubtractiveIMAC.

 

Fibrobactersuccinogenes
genenumber

Solubleproteinyield

w/oSUMOtag

w/SUMOtag

1425

0mg/liter

0mg/liter

1765

0mg/liter

10mg/liter

1793

0mg/liter

17mg/liter

1994

0mg/liter

17mg/liter

2201

0mg/liter

20mg/liter

Table1. ImprovementofsolubleproteinyieldwithSUMOtag.Yieldofsolubleproteinwasimprovedsignificantlyfor4of5FibrobactersuccinogenesgeneswhenclonedintopETiteN-HisSUMOandexpressedinHI-ControlBL21(DE3)Cells.Cultureswereinducedwith1mMIPTGandgrownovernightat22°C.Yieldswerecalculatedfromtheamountofpureproteinobtainedfrom100mlofcellcultureafterpurificationoveraNi-NTAcolumn.

CleavageofSUMOproteintag

AfterIMACpurificationoftheN-His-SUMOtaggedprotein,thetagportioncanberemovedpreciselybytheincludedSUMOExpressProtease.TheSUMOExpressProteaserecognizesthetertiarystructureofSUMOratherthanashortrecognitionsequenceandcleavespreciselyatthejunctionbetweentheSUMOtagandthetargetprotein,withnooff-targetcleavage.BoththeSUMOExpressProtease,whichis6xHistagged,andthecleavedN-His-SUMOtagcanthenbeseparatedfromthereleasedtargetproteinbysubtractiveIMAC.

Note:TheSUMOproteintagincludedinthesekitsisaspeciallyengineeredversionofSUMOthatcanbecleavedonlyusingLucigen'sSUMOExpressProtease.SUMOExpressProteasedoesnotcleavethewildtypeSUMOsubstrateatanyusefullevel.Therefore,itisnotrecommendedtouseSUMOExpressoProteasetocleavewildtypeSUMO.

ImportantProductUseInformation
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.

The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationof6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.

SUMOExpressProteaseismanufacturedandsuppliedbyLifeSensors,Inc.

ORDERINFORMATION

TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.

TheExpressoRhamnoseSUMOCloningandExpressionSystemcontainspre-processedpRhamN-HisSUMOVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.

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。


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