- Savetime!Enzyme-freecloninginsecondswithcloning-readyvectorandcells.
- Highefficiency:›90%recombinants.
- Tightly-controlledexpressionofN-orC-terminal6xHistaggedproteins.
AlsoavailablewithcleavableSUMOSolubilityTag
CompleteCloningandExpressionSystem
TheExpressoT7CloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenes.Thesystemsarecompletewithpre-processedpETite™N-orC-Hiscloningvectors,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidexpressionproblemsseenwithleakyT7promoter-basedsystems.TheN-orC-terminal6xHistaggedproteinscanberapidlypurifiedusingstandardNichromatography.SystemsarealsoavailablethatencodeanN-terminal6xHisSUMOtagfusionforimprovedsolubilityofexpressedprotein.OtherExpressoSystemsareavailablewhichexpressproteinsunderthecontroloftheRhamnosepromoter.
Five-SecondDirectionalCloningofPCR-AmplifiedGenes
TheExpressoT7CloningandProteinExpressionSystemusesExpressioneeringTechnology™,anenzyme-freerecombinationalcloningstrategytoseamlesslyintegratethegenewiththevector.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETite™T7expressionvector,andtransformimmediatelyintotheHI-Control™10GChemicallyCompetentCellsprovided(Figure1).
Cloning-ReadyVectorsSaveHoursofResearchTime.
NewpETiteT7vectorsinclude:
- StrongT7promoterforhigh-levelexpression.
- ChoiceofN-terminalorC-terminal6xHisfusiontagsforfastproteinpurification.
- Smallsize(2.2kb)foreasierdownstreammanipulation.
- PatentedCloneSmart®technologyincreasescloningefficiency.
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Figure2.pETiteT7expressionvectors:Smallsize(2.2kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Vectorsarepre-processedforinstantenzyme-freecloningofPCRproductswithachoiceofamino-terminalorcarboxyl-terminalfusionof6xHistagtoproteinofinterest. |
HighcloningefficiencywithHI-Control10GChemicallyCompetentCells.
ThehightransformationefficiencyofHI-Control10GChemicallyCompetentCellsensuresrecoveryofcloneswithprecisejunctionsandthecorrectorientation.Formostgenes,>90%ofcolonieswillhavethetargetgeneinsertedinthecorrectorientation.(Figure3).
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Figure3.HighcloningefficiencywithExpressoT7System. |
HI-ControlBL21(DE3)CellsControlLeakyProteinExpression
HI-ControlBL21(DE3)CellscontainhighlevelsoflacrepressortomaintaintightcontroloverexpressionofT7RNApolymerase.Tightercontrolmeansbettertoleranceofpotentiallytoxicgeneproducts(Figure4).
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Figure4.ExpressionofvariousproteinsusingtheExpressoT7SystemversusapETVector.GenesencodingaDNApolymerase,abluefluorescentprotein(BFP),orATPsynthasebsubunit(membraneprotein)wereclonedintopET28aorpETitevectorswithN-terminalorC-terminal6xHistags(asindicated).pET28aclonesweretransformedintostandardBL21(DE3)cells,andpETiteclonesweretransformedintoHI-ControlBL21(DE3)cellsforexpression.CulturesweregrowninLBat37°CtoanOD600of0.5to0.7(odd-numberedlanes)andinducedfor3hourswith1mMIPTG(even-numberedlanes).CellswerepelletedandlyseddirectlyinSDS-PAGEloADIngbuffer,and0.05ODequivalentswereanalyzedbygelelectrophoresis.ThegelwasstainedwithCoomassieblue. |
PurificationofActiveSolubleFluorescentProteinUsing6xHisTag
TheN-orC-terminal6xHistaggedproteinsexpressedusingtheExpressoT7CloningandExpressionSystemcanberapidlyaffinity-purifiedovercommerciallyavailablenickelresins.Anexampleofthepurificationofa6xHistaggedyellowfluorescentproteinclonedandexpressedusingtheExpressoT7SystemisshowninFigure5.
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Figure5.Purificationofa6xHistaggedfluorescentprotein.HI-ControlBL21(DE3)cellsharboringpETiteC-Hisvectorcontainingayellowfluorescentprotein(YFP)geneweregrownat37°CinLBmediatoanOD600of0.6(lane1),theninducedwith |
ImportantProductUseInformation:
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.
The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationofthe6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.
ORDERINFORMATION
TheExpressoT7Cloning&ExpressionSystemcontainspre-processedpETite®N-Hisand/orpETiteC-HisVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHI-ControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,andtransformationpositivecontrolpUCDNA.TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.
