Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

JournalofBiotechnology156(2011)245252ContentslistsavailableatSciVerseScienceDirectJournalofBiotechnologyjournalhomepage:/locate/jbiotecOverexpressionandpuricationoftherecombinantdiphtheriatoxinvariantCRM197inEscherichiacoliAlessandraStefana,b,MatteoContic,DiegoRubbolic,LorenzoRavaglia,EnricaPrestaa,AlejandroHochkoepplera,b,abcDepartmentofIndustrialChemistry,UniversityofBologna,VialeRisorgimento4,40136Bologna,ItalyCSGI,DepartmentofChemistry,UniversityofFlorence,ViadellaLastruccia3,50019Firenze,ItalyLaboratorioFarmaco-Tossicologico,AreaVastaRomagna,Cesena,ItalyarticleinfoabstractArticlehistory:Received30January2011Receivedinrevisedform20June2011Accepted15August2011Availableonline25August2011A.S.,M.C.,D.R.andA.H.dedicatethispapertothememoryofDr.SilvioBuzzi,whoalwaysenlightenedwithhissuggestionsthedevelopmentofthisproject.Keywords:Cross-reactingmaterial197SyntheticgeneOverexpressionEscherichiacoliPurication1.IntroductionTheexpressionoftherecombinantdiphtheriatoxinmutantCRM197inbacteriaotherthanCorynebac-teriumdiphtheriaehasproventobedifcult.Hereweproposeanewandalternativeprocedurefortheproductionoffull-lengthCRM197inEscherichiacoli.Thepresentstudyrelatesspecicallytotheexpres-sionofanarticialsequenceandtoamethodfortheisolationandpuricationofthecorrespondingprotein.Inparticular,asyntheticgenecodingforCRM197,bearingashorthistidinetagandoptimizedforE.colicodonusage,wasclonedinthepET9avector.Accordingly,theover-expressionofthepro-teinwassimplyinducedwitharabinoseinE.coliBL21AI.Therecombinantproteinwasinsolubleandalwaysfoundinsideproteinaggregates,whichweresolubilisedusingurea.Surprisingly,theexpressionofCRM197,devoidoftheshorttag,alwaysfailed.Followingarefoldingstep,thehis-taggedCRM197waspuriedbyafnityandgel-ltrationchromatographyandthepurityofthenalpreparationreached95%.Interestingly,therecombinantproteinfeaturesDNaseactivity,indicatingthatthepresenceofthetagisnotaffectingitsbiochemicalproperties.However,theremovalofthesynthetictagcouldbeeasilyobtainedbyincubatingthetargetproteinwithaproperquantityofacommercialenterokinase.2011ElsevierB.V.Allrightsreserved.thecell(Uchidaetal.,1973b).ThereisagrowinginterestinCRM197becauseofitspotentialantitumoractionrelatedtotheabilitytoTheCRM197proteinisavariantofthediphtheriatoxin(DTx,58kDa)characterisedbyasinglemutation(i.e.aglycine-glutamicacidsubstitutioninposition52)thatreducesitstoxicity(Uchidaetal.,1973a;Gianninietal.,1984).Theproteinnonethelessretainsthesameinammatoryandimmunostimulantpropertiesasthediphtheriatoxin,anditiswidelyusedasasafecarrierinconju-gatedvaccines(GuptaandSiber,1995;Shineeld,2010).Likethewild-typetoxin,CRM197comprisestwodomains,fragmentA(cat-alytic)andfragmentB,bondedtogetherbyadisulphidebridge.TheBdomaincontainsonesubdomainforthebindingtotheHB-EGFcellreceptorandanothersubdomainforthetranslocationinsideAbbreviations:CRM197,cross-reactingmaterial197;rCRM197,untaggedrecombinantcross-reactingmaterial;rCRM197his,his-taggedrecombinantcross-reactingmaterial;HB-EGF,heparinbindingepidermalgrowthfactor.Correspondingauthorat:DepartmentofIndustrialChemistry,UniversityofBologna,VialeRisorgimento4,40136Bologna,Italy.Tel.:+390512093671;fax:+390512093673.E-mailaddress:a.hochkoepplerunibo.it(A.Hochkoeppler).0168-1656/$seefrontmatter2011ElsevierB.V.Allrightsreserved.doi:10.1016/j.jbiotec.2011.08.024bindthesolubleformofHB-EGF,whichishighlyexpressedinsomehumancancers(Mitamuraetal.,1995;Buzzietal.,2004,2007).CRM197andothernon-toxicvariantshavealwaysbeenpro-ducedusinglysogenicculturesofCorynebacteriumdiphtheriae,infectedbyparticularphageswhosegenomecontainsamutatedversionofthetoxgeneencodingthediphtheriatoxin(Cox,1975;Rappuolietal.,1983;Rappuoli,1983).Theseproteinsaresecretedintotheculturemediumunderparticularconditions,thenrecov-eredbylteringorprecipitation,andsubsequentlypuriedusingchromatographicmethods(Cox,1975;Fassetal.,1995).How-ever,inordertoachievehighyieldsofCRM197,thefermentationprocessrequiresmultiplelysogenicmutantsofC.diphtheriaeandstrictlycontrolledconditionsofgrowth(temperature,agitation,aerationandferrouscontent).Undertheseoperatingconditions,theCRM197maximalconcentrationoccursafter20hofculture,thentherecoveryoftheproteindropsdramaticallyduetoprote-olysis(Rappuoli,1983).Ontheotherhand,fewstudiesconcerningtheuseofbacterialhostsalternativetoC.diphtheriaehavebeenreported.SeveralattemptshavebeenconductedinEscherichiacoli,althoughfocusedonlyontheexpressionoftruncatedformsof246A.Stefanetal./JournalofBiotechnology156(2011)245252Fig.1.SequenceofthesyntheticCRM197hisgene(rCRM197his);NdeIandBamHIrestrictionsites,usedforthecloningintothepET9avector,areshowninbold.Thesequencecorrespondingtothesynthetictag(28aa)isunderlined.TheGATGACGATGACAAAsequenceencodesfortheaminoacids(DDDDK)specicallyrecognisedbybovineenterokinase.ThecompletetagsequenceisMGGSHHHHHHGMASMTGGQQMGRDDDDK.Thistagwasproperlydesignedinordertobecutwithoutleavinganyextraresidues.CRM197,comprisingtheAdomainaloneand/orinassociationandtherCRM197hiscorrespondedtoapproximately80%ofthewithportionsoftheBdomain(Bishaietal.,1987a,b).Thesefrag-insolubleproteinfraction.HighlypuriedrCRM197hiswasnallyments,oftenproducedasfusionproteins,havebeenexpressedinobtainedaftertwochromatographicsteps,andthepureproteinE.coliusingdifferentpromotersandexpressionconditionswithmaintaineditsbiologicalproperties(e.g.DNaseactivity).lowyields(varyingfrom0.4to10mg/L;Bishaietal.,1987b).Ingeneral,thesestudieswereconductedinordertodeeplyinvesti-gatetheroleoftheAandBdomains(andtheirportions)interms2.Materialsandmethodsoftoxicity,bindingtothereceptor,proteinfoldingandstability(Spilsbergetal.,2005).Moreover,whiletheentireAdomainwas2.1.Bacterialstrainsandgrowthconditionsexpressedusingthenaturaltoxpromoter,theexpressionoftheBfragmentalonehasprovedverydifcultforitshighinstability,gen-erallyfollowedbyarapiddegradation(Cabiauxetal.,1988).TheexpressionofrecombinantDTxmutantshasbeenchallengedalsoinBacillussubtilisbyusingthesubtilisinsignalsequencefortheirsecretionintotheculturemedium(Zhou,1999).However,themax-E.coliBL21AIcellsFompTgaldcmlonhsdSB(rBmB)araB:T7RNAP-tetA(Invitrogen,Carlsbad,CA,USA)wereusedashostfortheexpressionofrecombinantCRM197.CulturesweregrowninLBstandardmediumsupplementedwithkanamycin(40g/mL).Arabinose(13mM,Sigma)wasaddedtoLBasinducer.imumyieldobtainedwasabout7.1mg/L.Arecentpaperdescribestheconstructionofahistidine-taggedversionoftheBdomainand2.2.CRM197genedesignitsproductioninE.coliinordertoevaluate,byELISA,theimmuneresponseinmiceandrabbitsagainstDTB(Nascimentoetal.,2010).ThesyntheticgenecorrespondingtoCRM197(EntelechonInthisstudy,thegenomicDNAofC.diphtheriaePW8strainwasGmbH,BadAbbach,Germany)wasclonedintopET9avectorusedastemplateforPCRamplicationofthedtbgenefollowedby(Novagen,Germany)usingNdeIandBamHIrestrictionsites.TheitscloningintoasuitableexpressionvectorforE.coli.TheDTBfrag-nucleotidesequencewasoptimisedforE.colicodonusageusingmentwasproducedwithanalconcentrationof0.38mg/mLandthesoftwareLeto(Entelechon).Twodifferentversionsofthegenewasrecognizedbyanti-DTantibodiesinwesternblottingassays.weredesigned:onesequencedevoidofanytag(rCRM197)andtheHence,theexpressionofthewholeCRM197proteininE.colisecondone(Fig.1)containing,atthe5end,anoligonucleotidehasnotbeenreported.Thepresentpaperprovidesamethodforsequence(84bp)encodingashorthistidinetag(rCRM197his).therapidproductionandpuricationoffull-lengthrCRM197(andCloningprocedureswereperformedfollowingstandardtech-itsderivatives)inE.coli,withcost-effectiveyields.Therecombinantniques.About20ngofeachrecombinantconstruct,pET9a-CRM197CRM197wasproducedasafusionproteinassociatedwithashortorpET9a-CRM197his,wereelectroporatedintoE.colicellsusingatageasilyremovabletoobtainthenativeform.UnderoptimizedGenePulserII(Bio-Rad,CA,USA)andtransformantswereselectedconditions,theexpressionyieldreachedupto40%oftotalproteinsonLBagarplatesupplementedwithkanamycin.A.Stefanetal./JournalofBiotechnology156(2011)245252247Fig.2.SDS-PAGEofproteinextractsobtainedfromE.coliBL21AI.(A)Sam-plesobtainedfromcellstransformedwithpET9a-CRM197.M:markersfeaturingmolecularmassesequalto116,66and45kDa.1:proteinsextractedfromnotinducedcultures;lanes24:samplesobtainedfrominducedcultures(1h,2h,ando/n,respectively).(B)SamplesobtainedfromcellstransformedwithpET9a-CRM197his.Lanes14:not-inducedorinducedsamples(sameconditionsasinA).ThearrowindicatesthebandcorrespondingtorecombinantCRM197his(approxi-mately60kDa).2.3.ProteinexpressionSinglecoloniesofBL21AIcontainingtherecombinantconstructs(pET9a-CRM197andpET9a-CRM197his)weregrownovernightat37Cin5mLofLBsupplementedwithkanamycin.Cultureswerediluted(1:500)infreshmediumandincubatedat37Cundershakingconditions.Theinducer(arabinose)wasthenaddedwhenpopulationsreachedanOD600equalto0.60.7.Aliquotswerehar-vestedbycentrifugation(4000gfor15min)atdifferenttimeintervalsafterinduction(1h,3handovernight).ToverifytheexpressionofrCRM197(withandwithoutthetag),totalproteinextractswereanalyzedbySDS-PAGE(10%acrylamide).Samplesweresimplypreparedaddingsamplebuffer5(10%SDS,10mM-mercaptoethanol,30%glycerol,0.2MTrisHClpH6.8,0.05%bro-mophenolblue)andboiledfor5min.Afterseparation,gelswerestainedwith0.1%CoomassiebrilliantblueR-250.2.4.ProductionFortheproductionofrecombinantCRM197his,200400mLofculturesweregrownunderthesameconditionsdescribedbefore.Cellswereharvestedbycentrifugationat4000gfor20minandpelletswereresuspendedin1/5ofvolumeoflysisbuffer(50mMFig.3.(A)KineticsofgrowthofculturesofE.coliBL21AIinLBmediumat37C.DiamondsrepresentE.coliBL21AIcontainingthepET9a-CRM197plasmidundernon-inducing(lled)orinducing(empty)conditions.CirclesindicateE.coliBL21AIculturescontainingthepET9a-CRM197hisplasmidundernon-inducing(lled)orinducing(empty)conditions.Arabinose13mMwasaddedtothecultures3haftertheinoculum(seearrow).(B)SDS-PAGEoftotalproteinextractsobtainedfromtheculturesdescribedabove.Lanes1and2:samplesfromnot-inducedandinducedBL21AI/pET9a-CRM197cultures,respectively;lanes3and4:samplesfromnot-inducedandinducedBL21AI/pET9a-CRM197hiscultures,respectively.TrisHClpH8,500mMNaCl,TritonX-1001%,phenylmethylsul-fonyluoride1mM).Cellularlysiswasobtainedbyshakingthesampleonicefor1hbeforecentrifugation.Alternatively,forsmall-scaleexperiments,cellsweresonicatedonice(815spulseswith15sintervals,at15WwithaSonicator3000,Misonix)andcen-trifugedfor20minat10,000g.Thesolublefractionwasremovedandpelletswereresuspendedinasolubilisationbuffercontaining50mMTrisHClpH8,500mMNaCl,TritonX-1001%and6Mureaat30Cfor3hinashaker.Solutionswerecentrifugedfor20minat10,000gandsupernatantscontainingtheinsolublefractionwerecollectedandstoredat4C.2.5.FirstpuricationstepSolubilisedsampleswereloadedintoametal-chelatingafn-itycolumn(5mL,Hi-TrapChelating,GEHealthcare).Non-specicbindingwasremovedbywashingwith10columnvolumesoftheequilibratingbuffer(50mMTrisHClpH8,500mMNaCl,1%Tri-tonX-100,6Murea),thenthedetergentwaseliminatedbeforestartingtherefoldingstep(eliminationofthedenaturantagent)andtheimidazolegradient(0500mM).Inparticular,ureawaseliminatedbyatwo-stepinversegradient(owrate0.5mL/min).ElutedfractionswereanalyzedbySDS-PAGE(10%acrylamide);samplescontainingrCRM197hiswerepooledanddialyzedagainst50mMTrisHClpH8,50mMNaCl.Theresultingsolutionwas248A.Stefanetal./JournalofBiotechnology156(2011)245252Tween20)toimprovetheefciencyandspecicityofthecleavage.Small-scaleexperimentswereperformedtodenetheappropri-ateratioofproteasetotargetprotein.Reactionswerestoppedbytheadditionofsamplebuffer(underreducingornot-reducingcon-ditions),followedbyheatingat95Cfor10min.SampleswereanalyzedbySDS-PAGE(15%acrylamide)todeterminetheextentofthecleavagecomparedtoundigestedsamplesascontrols.3.ResultsanddiscussionThepresentstudydescribesanalternativeprocedurefortheproductionoftheentirerecombinantCRM197providedwithashort,removable,N-tertag.Thisproteiniswidelyusedasacar-rierforconjugatedvaccines,sincenoformaldehydedetoxicationisrequiredanditsimmunogenicityisextremelyhigh.Moreover,CRM197presentsgreatpotentialasananti-tumoragentandasasafedrugagainstatherosclerosis(bothcancerandvascularplaquesoverexpressHB-EGF,specicreceptorforCRM197;Buzzietal.,2007;Buzzi,2009).HencetheevidentneedtodisposeofamethodfortheproductionofthewholeCRM197withcost-effectiveyields,reducedtime,andsimpleculturemediumusingE.coliasbacterialhost.3.1.ExpressionofrecombinantCRM197ThesequencecorrespondingtothewholeCRM197,devoidofFig.4.SDS-PAGEofrecombinantCRM197his.(A)Lanes1and2:solubleproteinthenaturalsignalsequencefortheexportationoutsidethecell,fractionfromanot-inducedor3h-inducedcultures,respectively.Lanes3and4:wasusedtoobtainanarticialsequenceoptimisedfortheexpres-insolublefractionobtainedfromthesamesamplesaftertheremovalofsolubleproteins.M:molecularmassmarkers.Thearrowshowsthebandcorrespond-ingtorCRM197his.(B)DifferentsolubilizationbufferstestedfortherecoveryofrCRM197hisfromtheinsolublepellet.Lanes1and2:50mMTrisHClpH8,500mMsioninE.coli.Thedesignedgenewasassociated,atthe5-terminalend,withanoligonucleotidesequencecodingforahexa-histidinestag,allowingafnitychromatography(Fig.1).ThisarticialgeneNaCl,0.51%TritonX-100;lanes3and4:50mMTrisHClpH8,500mMNaCl,wasclonedintothepET9avectorundertheT7promoter,and0.51%SDS;lanes5and6:50mMTrisHClpH8,500mMNaCl,0.51%Tween20.Solubilizationwasperformedundershakingconditionsat30Cfor3h.theresultingconstructwassubsequentlyintroducedintoE.coliBL21AI,bearingthechromosomalaraBADpromotertocontroltheconcentratedbyultraltration(AmiconcellwithYM-10mem-brane,Millipore,MA,USA)andproteinconcentrationwasthendeterminedbytheBradfordassay(BioRadProteinAssay).T7RNApolymerasemessengersynthesis.Alternatively,thesamesyntheticgene,withoutanytag,wasclonedintothesamevector.Theexpressionlevel,followingarabinoseinduction,wascheckedbySDS-PAGEwherethetargetproteinwasexpectedtorunasa2.6.Secondpuricationstepbandofabout5861kDa,dependingonthepresenceofthetag.Dif-ferentconditionsofgrowth(lengthofinductiontime,temperature,TheresultingconcentratedrCRM197hisfractionswereappliedtoaSuperdex-200gelltrationcolumn16/70(GEHealthcare,UK)equilibratedwith50mMTrisHClpH8,50mMNaCl(owrateof0.6mL/min).ThepresenceofrCRM197hisanditspuritylevelintheelutedfractionswasevaluatedbySDS-PAGEfollowedbyaSilverstainingprocedure(PageSilverTMKit,FermentasGMBH,Germany).inducerconcentration)werealsoanalyzedinordertoidentifyanoptimisedprotocolyieldingthemaximumexpression.Despitealltheconditionstested,thebandcorrespondingtotherCRM197(notag)wasneverdetectedingels(Fig.2A),whereashighexpressionlevelofrCRM197hiswasalwaysobtained,asshowninFig.2,whereadistinctbandofabout61kDaisclearlyvisible(Fig.2B).Thisobser-vationsuggeststhatrCRM197,devoidofthetag,istoxicforE.coli2.7.Nucleaseactivityassayor,alternatively,thattheproteinisextremelyunstableandeasilydegraded.Thenucleaseactivitywa