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Lucigen/Expresso® Rhamnose Cloning and Expression System, C-His/49012-2/10 rxns
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CloneaPCRamplifiedgeneinaneffortlessafternoon,andexpressrecombinantproteinthenextday.Five-second,directional,enzyme-freePCRcloning!90%recombinants!Singlecompetentcellhoststrainforbothcloningandexpression.TighterexpressioncontrolwithtunableRhamnose(rhaPBAD)promoter.Highthroughputformatfriendlywithautoinductionreagents.AlsoavailablewithcleavableSUMOSolubilityTagCompleteCloningandExpressionSystemswithExpressioneering™TechnologyTheExpressoRhamnoseCloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenesusingExpressioneeringTechnology.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCRamplifiedDNAintospeciallydesignedexpressionvectorswithouttheneedforenzymesorpurificationsteps.Asinglehoststrainisusedforbothstablecloningandcontrolledproteinexpression,makingExpressoRhamnosethefastestcloningandexpressionsystemsavailable.Thesystemscomecompletewithpre-processedexpressionplasmidsandcompetentcells,suppliedinsingletransformationvials.Figure1.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCRamplifiedDNAintospeciallydesignedexpressionvectorswithouttheneedforenzymesorpurificationsteps.Thedesiredinsertissimplyamplifiedwithprimersthatinclude18basesthatoverlapwiththeendsoftheExpresso®vector.TheunpurifiedPCRampliconisthenmixedwiththepRhamexpressionplasmidandthehigh-efficiencycompetentcellsprovided,anddirectlyplatedonappropriatemedia.TheExpressoRhamnoseSystemsutilizetherhamnose-inducIBLerhaPBADpromoterfortightcontrolofproteinexpression.TranscriptionfromtherhaPBADpromotercanbe“tuned”usingdifferentconcentrationsofrhamnosetoidentifytheoptimalexpressionlevelsfordifficulttargetproteins.Simpleautoinductionprotocolsusingrhamnoseandglucosesolutionssuppliedwiththekitsallowproteinexpressionwithminimalintervention.ThepRham™N-HisandpRhamC-HisVectorsprovidedintheExpressoRhamnoseCloningandExpressionSystemfacilitateinstantcloningoftargetgeneswithachoiceofamino-orcarboxyl-terminal6xHisaffinitytags.The6xHispeptideprovidesforfastandeasyaffinitypurificationofproteinsundernativeordenaturingconditions.ForenhancedsolubleproteinexpressionusingSUMOfusiontagtechnology,seetheExpressoRhamnoseSUMOSystem.WithinstantcloningusingExpressioneeringTechnology,asingle-hostsystemforcloningandexpression,andsimpleautoinductionprotocols,theExpressoRhamnoseSystemsarehighlyamenabletohigh-throughputcloningandproteinexpression.  Figure2.pRhamexpressionvectors.RBS,ribosomebindingsite;ATG,translationstartsite;Stop,translationendsite;Kan,kanamycinresistancegene;ROP,RepressorofPriming(forlowcopynumber);Ori,originofreplication.CloneSmart®transcriptionterminators(T)preventtranscriptionintooroutoftheinsert,andaterminatorfollowsthecloningsite.The6xHisaffinitytagisfusedtotheaminoterminus(pRhamN-His)oratthecarboxylterminus(pRhamC-His)oftheexpressedtargetprotein.Alsoavailable:pRhamN-HisSUMOVectorforenhancedsolubleproteinexpressionwithcleavableSUMOsolubilitytag. pRhamExpressionVectorsThepRham™N-HisandpRhamC-HisVectors providedintheExpressoRhamnoseCloningandExpressionSystemareshowninfigure2. LikethepETiteVectorsfeaturedintheExpressoT7kits,thepRhamVectorsusedintheExpressoRhamnosekitsarebasedonthepSmartvectorbackbone,whichfeaturespatentedCloneSmart®technologyforincreasedcloningefficiency.Thepre-processedpETiteandpRhamvectorsfeaturethesamesequencesflankingthecloningsite,allowingasinglePCRproducttobeclonedintoeithervector.Single-HostcloningandtunableexpressionwithExpressoRhamnoseSystemFigure3.Tuningrecombinantproteinexpressionlevelswithrhamnoseinduction.ThepRhamC-HisKanVectorcontainingageneencodingabluefluorescentprotein(BFP)wastransformedintoE.cloni10Gcells.AnuninducedstarterculturewasinoculatedtoastartingOD600of0.8intoculturetubescontainingLBmediawith30µg/mlkanamycinandtheindicatedconcentrationsofrhamnose(0to0.2%w/v)or2%glucose.Afterovernightincubationat37°C,sampleswereharvestedbycentrifugation,lysedinSDS-PAGEloADIngbuffer,andanalyzedbySDS-PAGE.TheCoomassie-bluestainedgelshowstotalcellularprotein.Proteinexpressionlevelsareresponsivetorhamnoseconcentrationsbetween0.001%and0.2%. TherhaPBADpromoteristightlyshutoffintheabsenceofthesugarrhamnose,allowingtheuseofasinglehoststrainforbothcloningandproteinexpression.ThelevelofinductionfromrhaPBADisresponsivetodifferentconcentrationsofrhamnose(Fig.3),allowingyoutotunethelevelofexpression,especiallyforproteinsthataretoxicorinsolublewhenoverexpressed.MaximalproteinexpressionfromtherhaPBADpromoteristypicallylowerthanfromtheT7promoter,butcanstillreachveryhighlevels(upto100mg/l). Figure4.AutoinductionofproteinexpressionwiththeExpressoRhamnoseSystem.FlasksofLBmediacontaining30 µg/mlkanamycin,0.2%rhamnose,andeither 0.05%glucose(earlyautoinduction,upperpanel)or0.15%glucose(lateautoinduction,lowerpanel)wereinoculatedtoaninitialOD600of0.4withanuninducedcultureofE.cloni10GcellsharboringtheT4ligasegeneinthepRhamN-HisVector.SamplesofthecultureswereharvestedattheindicatedtimepointsforSDS-PAGEanalysis.Inductionofligaseexpressionbeganby4hoursintheearlyautoinductionculture,or8hoursinthelateautoinductionculture.BothculturesreachedsimilarOD600by24hours.  Figure5.Directautoinductionofrecombinantproteinexpressionfromindividualcolonies.ThepRhamC-HisVectorcontainingtheBFPgenewastransformedintoE.cloni10GcellsandplatedonYTagarplatescontaining30µg/mlkanamycin.SinglecolonieswerepickedfromtheplateandinoculateddirectlyintoLBliquidmediacontainingkanamycin(30µg/ml),rhamnose(0.2%w/v),andeither0.05%(earlyautoinduction)or0.15%(lateautoinduction)glucose.SampleswereharvestedatthetimepointsindicatedandanalyzedbySDS-PAGE.ConvenientAutoinductionwithExpressoRhamnoseSystemsTranscriptionfromtherhaPBADpromoterissubjecttorepressionbyglucose.Whenbothglucoseandrhamnosearepresent,glucoseismetabolizedpreferentiallyandtherhaPBADpromoterremainsinactive.Upondepletionofglucose,therhaPBADpromoterisactivatedbyrhamnose.Convenientautoinductionprotocolsuseacombinationofglucoseandrhamnosetoallowinductionofproteinexpressionwithminimalintervention.Inoculatefromastarterculture(Fig.4)ordirectlyfromindividualcolonies(Fig.5)intoautoinductionmedia,andinductionoccursautomatically.Thetimingofinductioncanbeadjustedwiththeuseofdifferentglucoseconcentrations.SolutionsofrhamnoseandglucoseareprovidedwiththeExpressoRhamnosekits.SeeApplicationNoteinNatureMethods,"Expresso®CloningandExpressionSystems:Expressioneering™Technologystreamlinesrecombinantproteinexpression."Alsoavailable:ExpressoRhamnoseSUMOSystemwithcleavable6xHisSUMOtagforenhancedsolubilityofexpressedproteins. 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.ORDERINFORMATIONTheExpressoRhamnoseCloning&ExpressionSystemcontainspre-processedpRhamN-Hisand/orpRhamC-HisVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,transformationpositivecontrolpUCDNA,andforwardandreversePCRprimerstoconfirmclones.TheExpressoRhamnoseSUMOCloningandExpressionSystemcontainspre-processedpRhamN-HisSUMOVectorDNA,single-transformationE.cloni10GChemicallyCompetentCells(SOLOs),andtheauto-inductionreagents20%Rhamnosesolutionand15%Glucosesolution.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.

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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