CloneaPCRamplifiedgeneinaneffortlessafternoon,andexpressrecombinantproteinthenextday.

  • Five-second,directional,enzyme-freePCRcloning!90%recombinants!
  • Singlecompetentcellhoststrainforbothcloningandexpression.
  • TighterexpressioncontrolwithtunableRhamnose(rhaPBAD)promoter.
  • Highthroughputformatfriendlywithautoinductionreagents.

AlsoavailablewithcleavableSUMOSolubilityTag

CompleteCloningandExpressionSystemswithExpressioneering™Technology
TheExpressoRhamnoseCloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenesusingExpressioneeringTechnology.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCRamplifiedDNAintospeciallydesignedexpressionvectorswithouttheneedforenzymesorpurificationsteps.Asinglehoststrainisusedforbothstablecloningandcontrolledproteinexpression,makingExpressoRhamnosethefastestcloningandexpressionsystemsavailable.Thesystemscomecompletewithpre-processedexpressionplasmidsandcompetentcells,suppliedinsingletransformationvials.

Expressioneering Protein Expression

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. 

 
pRham Vectors

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.

 

pRhamExpressionVectors
ThepRham™N-HisandpRhamC-HisVectors providedintheExpressoRhamnoseCloningandExpressionSystemareshowninfigure2. LikethepETiteVectorsfeaturedintheExpressoT7kits,thepRhamVectorsusedintheExpressoRhamnosekitsarebasedonthepSmartvectorbackbone,whichfeaturespatentedCloneSmart®technologyforincreasedcloningefficiency.Thepre-processedpETiteandpRhamvectorsfeaturethesamesequencesflankingthecloningsite,allowingasinglePCRproducttobeclonedintoeithervector.

Single-HostcloningandtunableexpressionwithExpressoRhamnoseSystem

Tuning Recombinant protein expression

Figure3.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).

 
Autoinduction of protein expression

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.

  
Direct autoinduction of recombinant protein

Figure5.Directautoinductionofrecombinantproteinexpressionfromindividualcolonies.ThepRhamC-HisVectorcontainingtheBFPgenewastransformedintoE.cloni10GcellsandplatedonYTagarplatescontaining30µg/mlkanamycin.SinglecolonieswerepickedfromtheplateandinoculateddirectlyintoLBliquidmediacontainingkanamycin(30µg/ml),rhamnose(0.2%w/v),andeither0.05%(earlyautoinduction)or0.15%(lateautoinduction)glucose.SampleswereharvestedatthetimepointsindicatedandanalyzedbySDS-PAGE.

ConvenientAutoinductionwithExpressoRhamnoseSystems
TranscriptionfromtherhaPBADpromoterissubjecttorepressionbyglucose.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.

ORDERINFORMATION

TheExpressoRhamnoseCloning&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.