TheExpresso®CMVCloningandExpressionSystemcombinesinstant,enzyme-free,directionalcloningwitha“noscar”vectorforeukaryoticgeneexpression.ThiscloningsystemisbasedonExpressioneering™Technology,whichusesinvivohomologousrecombinationtoseamlesslyclonePCR-amplifiedDNAintospeciallydesignedexpressionvectorswithoutpurificationorenzymaticincubations.It’sthefastest,easiestmammalianexpressionsystemavailable.

Figure1.ExpressioneeringTechnologyusesinvivohomologousrecombinationtoseamlesslyclonePCR-amplifiedDNAintothepME-HAvector.Targetgenesareamplifiedwithprimersthatinclude18basesofoverlapwiththeendsoftheExpressovector.TheunpurifiedPCRampliconissimplymixedwiththepME-HAexpressionplasmidandimmediatelytransformedintothehighlycompetentcellsprovided.NoDNApurification,enzymetreatment,vectorpreparation,orligationstepsarerequired.

pME-HAVector

TheExpressoCMVCloningandExpressionSystemfeaturesthepME-HAexpressionvector,whichcontainsalltheelementsyouneed,andnothingyoudon't.TheCMVpromoterenablesstrong,constitutiveexpressioninthesmallestmammalianexpressionvectoravailable.The3.4kbvectorsupportshighertranfectionefficiencyandeasierdownstreammanipulation.CombinedwithExpressioneeringtechnologyforinstant,scar-freecloning,theresultissurprisinglysimplemammalianexpression.

Figure2.pME-HAexpressionvectorTheCMVpromoterdrivesexpressionofthePCRinsert;ampandSV40promotersexpressthegeneforkanamycin/neomycinresistanceinbacteriaandmammaliancells.ThepUCorigingiveshighplasmidyields.CloneSmart®transcriptionterminators(T)preventtranscriptionintooroutofthevectorbackbone,increasingclonestABIlity.TheHAaffinitytagcanbefusedtothecarboxyterminusoftheexpressedprotein,ifdesired.

StrongExpression

ThepME-HAvectorprovidesrobustproteinexpression,greaterthanorequaltothatofthecommonly-usedvectorpCDNA3.1.ProteinexpressionfromtheGFPgene(0.8kb)andthefulllengthβ-galactosidasegene(3kb)wasdetectedbyWesternblottingagainsttheHAtaginCHO-K1andCOS-7transfectants(Figure3).β-galactosidaseexpressionwasalsomeasuredquantitativelyintransfectedCHO-K1cells(Figure4).

Figure3.ComparisonofproteinexpressionfrompME-HAandpcDNA3.1vectorsinCHOandCOScells.CHOandCOScellsweretransfectedwithconstructsencodingGreenFluorescentProtein(GFP)andbeta-galactosidase(β-gal)usingLipofectamine2000(Invitrogen)followingthemanufacturer'srecommendation.At48hposttransfection,wholecelllysateswerefractionatedbySDSPAGE,transferredtoaPVDFmembrane,anddetectedwithanti-HAantibody.ExpressionfromthepME-HAvectorwasequaltoorgreaterthanthatfromthe“standard”pcDNA3.1vector.

Figure4.CHO-K1cellsweretransfectedwithpME-HA-Bgal plasmidataratioof2:1(µlTransIT-2020transfectionreagent:µgDNA).CellswereassayedforBgalactivity24-hourspost-transfectionusingtheMammalianB-GalactosidaseAssayKit(ThermoScientific,#75707),andreadat405nmonaplatereader.Absorbancevaluesweredividedbybackgroundabsorbance(mock-transfectedcells).

ExpressionofDifficultProteins

TheuseofExpressioneeringtechnologyresultsinprecisecloning,withouttheadditionofunwantedsequence(suchasrestrictionsites)typicallypresentinmultiplecloningsites.TheminimalsizeofthepME-HAvectoralsocontributestoenhancedcloningcapability.NumerousGPCRgeneshavebeenclonedandexpressedinmammaliancellsusingtheExpressoCMVsystem.WesternblottingwasusedtodetectexpressionoftheHA-taggedGPCRs(Figure5),andfunctionalitywasconfirmedbyrADIoactiveligandbindingassays(Figure6).

Figure5.ExpressionofGPCRsfromthepME-HAvector.COS-7cellsweretransfectedwithconstructsencodingturkeybeta1-adrenergicreceptor(β1-AR)andhumanadenosineA1receptor(A1A)withTransIT-2020(MirusBioLLC)orLipofectamine2000(Invitrogen)followingmanufacturer'srecommendation.At48hourspost-transfection,wholecelllysateswerefractionatedbySDS-PAGE,transferredontoPVDFmembrane,anddetectedwithanti-HAantibody.

Figure6.RadioligandbindingdataforGPCRsexpressedfromthepME-HAvector.Cellsexpressingb1-ARwereanalyzedforradioligandbindingusing20nM[3H]-Dihydroalprenolol(Perkinelmer).Non-specificbindingwasdeterminedinpresenceofexcessunlabeledcompetitor,10μM(S)-(-)-Propanololhydrochloride.ReceptorboundligandwascapturedonGF/Bfiltersandcounted.CellsexpressingA1Areceptorwereanalyzedbybindingof10μM[3H]-DPCPX(PerkinElmer).Non-specificbindingwasdeterminedinpresenceofexcessunlabeledcompetitor,10μM8-Cyclopentyl-1,3-dipropylxanthine.BoundligandwascapturedonGF/Bfiltersandcounted.