FinalThesis

Developmentofanexpressionsystemforadehydrogenase

AxelVeibäckLITH‐IFM‐A‐EX‐‐10/2235‐‐SE

DepartmentofPhysics,ChemistryandBiologyLinköpingUniversitySE‐58183Linköping

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Abstract Inrecentyears,biocatalyticalstepsinchemicalsynthesisarebecomingincreasinglyimportantforeconomicalandenvironmental‐friendlyproduction.InordertoevaluatetheuseofenzymesinaprocessatCambrexKarlskogaAB,anexpressionsystemwasdevelopedforadehydrogenase.AsyntheticgenewasclonedintoEscherichiacoliDH5αcells,usingthepTZ19Rexpressionvector,aspreviouslydescribedintheliterature.Proteinexpressionwascarriedoutat25°C,30°Cand37°CandresultsweremeasuredusingSDS‐PAGEandactivityassays.Toimproveexpression,thegenewasmodifiedinthreewaysusingPCR,yieldingeightclones:ItwasinsertedintothepSE420expressionvector,shortenedtoavoidinclusionbodyformationandamissingnucleotidewasinsertedintothesequence.Aprotocolforinclusionbodyscreeningwasalsodeveloped.Finally,anassayfordeterminingthekineticconstantsofdehydrogenasewasdesigned.Itisconcludedthatfurtherexperimentsmustbedonetoobtainexpressionofthedehydrogenaseandrecommendationsforadditionalworkaregiven.

Keywords:biocatalysis,proteinexpression,geneexpression,inclusionbodies,SDS‐PAGE,polymerasechainreaction,pTZ19R,pSE420

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Sammanfattning Biokatalytiskaprocessteghardesenasteårenblivitettalltviktigareinslagikemisksyntesförattåstadkommaekonomiskochmiljövänligproduktion.FörattutvärderaanvändandetavenzymerienprocesshosCambrexKarlskogaAButveckladesettexpressionssystemförettdehydrogenas.EnsyntetiskgenklonadesiniEscherichiacoliDH5αochuttrycktesmedhjälpavexpressionsvektornpTZ19R,somtidigarefinnsbeskrivetilitteraturen.Proteinuttrycketutfördesvid25°C,30°Coch37°CochresultatetmättesmedhjälpavSDS‐PAGEochaktivitetsmätningar.Genenfördehydrogenasetmodifieradespåtresätt,vilketgavupphovtillåttavarianter.GenenfördesövertillexpressionsvektornpSE420,kortadesförattundvikabildningavinklusionskropparochennukleotidsomfattadesfrångensekvensenåterinfördes.Ettprotokollutarbetadesävenförundersökningavinklusionskroppar.Tillsistsammanställdesenmetodförattundersökadekinetiskakonstanternahosdehydrogenaset.Slutsatsenavarbetetärattfortsattastudiermåsteutförasföratterhållauttryckavdehydrogenasetochrekommendationergesförframtidaundersökningar.

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A note on secrecy Thisthesisispublicwork.However,forthistobepossibleandnotpresentadisadvantageforCambrexKarlskogaAB,partsofthisworkthatcouldbeusedtoidentifytheexactprocesshavebeenomitted.Thisincludesthenameofthesubstrateusedandproductformedandthenameandmolecularweightoftheenzymecentraltothisreport,aswellasliteraturereferencesdealingwiththedehydrogenase.However,theprocessandmethodsofdevelopingtheexpressionsystem,togetherwithresultsanddiscussionthereofhavenotbeenleftout.

Theenzymeusedwillbereferredtoasdehydrogenasethroughoutthisreport.

Thesubstrateandproductwillbereferredtoassubstrateandproduct.

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Abbreviations ADH Alcoholdehydrogenase

dH2O Deionisedwater(MilliQwater)

DNA Deoxyribonucleidacid

EDTA Ethylenediaminetetraaceticacid

HPLC Highperformanceliquidchromatography

IPTG Isopropylβ‐D‐1‐thiogalactopyranoside

LB Lysogenybroth

LB/Amp Lysogenybrothwithampicillin

NADP Nicotinamideadeninedinucleotidephosphate(oxidizedform)

NADPH Nicotinamideadeninedinucleotidephosphate(reducedform)

PCR Polymerasechainreaction

PMMA Polymethylmethacrylate

PMSF Phenylmethylsulfonylflouride

SDS‐PAGE Sodiumdocecylsulfatepolyacrylamidegelelectrophoresis

SOB Superoptimalbroth

SOC Superoptimalbrothwithcataboliterepression

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Table of contents Abstract iSammanfattning iiAnoteonsecrecy iiiAbbreviations ivTableofcontents v1 Introduction 12 Theoreticalbackground 22.1 Biocatalysis 22.2 Dehydrogenases 22.3 Thesoughtreaction 32.4 Coenzymeregeneration 32.5 Reactionmedium 42.6 Geneexpression 42.6.1 Recombinantgeneexpression 42.6.2 Controlofgeneexpression 42.6.3 T7Expressionsystem 52.6.4 Expressionvectors 52.6.5 ComplicationswithE.coli 62.6.6 Inclusionbodies 72.6.7 Proteolysis 7

2.7 Experimentalmethods 72.7.1 Restrictionendonucleases 72.7.2 Ligation 82.7.3 Polymerasechainreaction 82.7.4 Geneticengineering 9

2.8 Kineticcharacterization 103 Experimentaldetails 113.1 Cloning 113.1.1 Restrictionendonucleasecleaving 113.1.2 DNAseparation 113.1.3 Concentrationmeasurements 123.1.4 Ligation 123.1.5 Transformation 12

3.2 Analyticalmethods 123.2.1 Plasmidpreparation 123.2.2 Sequencing 123.2.3 Cellcultivation 133.2.4 Celllysis 133.2.5 Proteinseparation 133.2.6 Enzymeactivityassays 133.2.7 Inclusionbodyscreening 14

3.3 DNAmodifications 143.3.1 Implementedmodifications 143.3.2 Polymerasechainreaction 153.3.3 CloningofmodifiedDNA 15

3.4 Kineticcharacterization 16

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4 Resultsanddiscussion 174.1 Proteinexpression 174.2 Kineticcharacterization 21

5 Conclusions 246 Recommendations 257 Acknowledgements 268 References 27Appendix:Growthmedium 29

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1 Introduction Theuseofbiocatalyticalmethodsfortheproductionofchemicalsandpharmaceuticalsarebecomingincreasinglypopular.CambrexKarlskogaABhasahistoryofmanufacturingchemicalsthatdatesbacktothelaboratoriesofAlfredNobelinthelate19thcentury.Thecompanysupportsthepharmaceuticalindustrybymanufacturingactivepharmaceuticalingredients,andprovidesresearchanddevelopmentfortheirclientsaswellasin‐houseproducts.Duringthelastdecadesinitiativeshavebeentakentofurtherinvestigatetheuseofbiocatalyticmethodsinthefabricationofchemicalproducts.ThebiocatalystdepartmentatCambrexwasmovedtoKarlskogain2008.

ForanewproductatCambrex,biocatalyticpathwaysarebeingconsideredformanufacturing.Aninterestingenzyme,previouslyreportedintheliterature,wastargetedandinvestigated.Asyntheticgenewasordered,basedonsequenceinformationfromtheliterature.TheenzymehaspreviouslybeenmanufacturedinEscherichiacoliDH5αcells,usingpTZ19Rexpressionvectors,butproductiondetailsarenotavailable.Inordertocompleteandevaluateaproductionprocess,anexpressionsystemfortheenzymemustbesetup.

Theaimofthisprojectistodevelopandoptimizeanexpressionsystemforaspecificdehydrogenaseenzyme.Thisincludescloningthegeneintoasuitablehost,optimizingexpressionconditionsandmeasuringenzymeactivity.

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2 Theoretical background

2.1 Biocatalysis Inrecentyears,biocatalyticproductionofchemicalshasdevelopedintoawidelyusedmanufacturingmethod.Itcanhaveseveraladvantagesovertheconventionalchemicalapproachforproduction,mostsignificantlytheselectivitiesoftheenzymesusedandthemildoperationalconditionsofthereactions[Panke,2004].Protectiveblockinganddeblockingstepsofotherlabilefunctionalgroupsonthesubstratecanbeeliminatedthankstothesite‐specificenzymes,aswellasreactionsdemandinghighpressureandhightemperatures.Thismeansthatbiocatalysisinmostcasesisconsideredamoreeconomicalandenvironmental‐friendlymethodthanthechemicalroute[Ishige,2005].Thisiswidelyseenasapromisingresearchfieldandmanyinitialproblemstothetechniquesuchastheavailabilityofbiocatalystsandtheiroperationalstabilityhavebeenhelpedthroughmolecularbiologymethods,suchasdirectedevolution[Schoemaker,2003].Somedrawbacksofbiocatalysisarethatsinceeachenzymeisrelativelyspecific,amatchingenzymethatproducestheproductinasatisfyingyieldmustbefound,whichcanprovetediouswork.Theenantiospecificnatureofenzymesisnotalwayspositive,andacompletelydifferentenzymeisusuallyneededtocoverbothformsofasubstrate.Enzymesworkbestinwater,whereaswaterisusuallynotsuitablefororganicreactions.Enzymaticreactionsinothersolventswillusuallywork,butwithsomelossofactivity[Faber,2000].Advancedchemicalroutesarelikelytoremainimportantinthefuture,butacombinationofchemicalroutesandbiocatalysiswheresuitablewillprobablybemorecommon[Schoemaker,2003].

Biocatalysiscanbecarriedoutwithisolatedenzymesorwholecellsystems.Usingwholecellcatalysiscanbebotheasierandmoreinexpensive.Enzymesarekeptintheirnaturalsurroundingsandaregenerallymorelong‐termstablethanfreeenzymes[Ishige,2005].Whetherwholecellsystemscouldbeuseddependsonmanyfactors,themostimportantbeingtypeofreaction,cofactorrecyclingneedsandscaleofbiocatalysis[Faber,2000].However,reactionsaremoreunpredictableduetosidereactionsandthemorecomplexcomposition.Usingwholecellsystemsinanindustrialscalecouldprovedifficult[Ischige,2005].

2.2 Dehydrogenases Theenzymesinvolvedinoxidationandreductionofbiologicalcompoundsarecalledoxidoreductases[Botham,2009].Oxidoreductasesaccountforaboutonefourthofallknownenzymes[Liu,2007].Theyareclassifiedintofourgroups:oxidases,dehydrogenases,hydroperoxidasesandoxygenases.Thisstudyfocusesonenzymesfromthesecondgroup,thedehydrogenases.Dehydrogenaseshelptransferhydrogenbetweenasubstrateandacoenzymeorhydrogencarriers.Thesearetypicallynicotinamidecoenzymesorriboflavin[Botham,2009].Nicotinamideadeninedinucleotide(NAD)andNicotinamideadeninedinucleotidephosphate(NADP)arethetwomostcommonlyencounteredcoenzymes.Theenzymegroupiscapableoftransformingprimaryandsecondaryalcoholstoaldehydesandketonesandprimaryaminestoprimaryimines.Dehydrogenasesusingflavinasacofactorarealsocapableofintroducingadoublebondbetweentwocarbonatoms[Bugg,2004].Dehydrogenaseshavefoundwidespreaduseinclinicalandfoodanalysis[Hummel,1989]andinprocessessuchassynthesisofchiralcompounds,preparationandmodificationsofpolymers,biosensorsanddegradationoforganicpollutants[Liu,2007].

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2.3 The sought reaction Thesoughtreactionistooxidizeasecondaryalcoholtoaketoneusingthedehydrogenase.TheenzymeusedrequiresNADPasacoenzyme(Figure1).

Thesubstrateusedisreadilyavailable,butcontainslabilefunctionalgroups.Bybeingabletoperformthisreaction,aloneorincombinationwithchemicalmethodsthataddressesotherfeaturesinthesubstrate,theeconomicalvaluecanbegreatlyincreased.Usinganenzymeforthereactionwilladdressaspecifichydroxylgrouponthesubstrate,leavingotherfunctionalgroupsuntouched.

Generally,thereversereactionwhereketonesarereducedismorepopular.Astudyinvestigating205selectedpatentsintheareaofbiocatalysisandbiotransformationbetween2000and2004revealed35patents(or17%)focusingonketonereductionwhereasthereversereactionwasrare[Panke,2004].

Figure1:Thereactionoftheexamineddehydrogenase:AsecondaryalcoholisoxidizedtoaketonewiththehelpofthecoenzymeNADP.

2.4 Coenzyme regeneration Adrawbackwithbiocatalysisisthatcofactor‐dependentenzymesarealmostexclusivelytiedtotheirnaturalcofactors,inthiscaseNADP[I].Thesecofactorsaregenerallyexpensiveandrelativelyunstableandcannotbereplacedbymoreeconomicalman‐madesubstitutes.Thishasledtothatinsyntheticreactionsdemandingcofactors,asubstoichiometricamountisoftenusedandinstead,acoupledreactiondemandingthecorrespondingoxidizingorreducingagentisusedtoregeneratetheoriginalcofactor.Thisrecyclingisnotatrivialtask,despiteprogressesmadeinthefield[Faber,2000].Traditionally,reactionshavebeenperformedusingwholecellsystems,wheresomerecyclingisperformednaturally,butwiththeincreasinginterestofimmobilizedenzymaticreactionssincethelate1990’s,newwaysofcoupledreactionsarebeinginvestigated[Liu,2007].

[I]Nicotinamideadeninedinucleotidephosphate(NADP)

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2.5 Reaction medium Thereareseveralfactorsthatneedtobeconsideredwhilesettinguptheenzymaticreaction:temperature,reactantconcentrations,theformoftheenzyme,suitablesolvent,levelofresidualwaterandacid‐baseconditions[Halling,2002].

Generally,enzymesarethemostefficientinaqueoussolutions,butthisisnotalwaysthebestchoice.Sincebiotransformationsoftenincludemoleculeswithalowsolubilityinwater,reactionscouldworkbetterinsomeorganicmediaorasolventwithalowamountofwater[Schoemaker,2003].Itisthereforeoftenpreferredtocarryoutthereactioninanon‐aqueousmedium.Reasonsbehindthisisthatreactantsmightnotbesolubleinwater,equilibriaandkineticsofthereactioncouldbechangedduetothesolvent,differentmediacouldbeusedtotunespecificity,side‐reactionsandmicrobialgrowthinaqueousmediacanbeavoidedandtheprocessisbetterintegratedtothechemicalstepsofthereaction[Halling,2002].

Enzymesinaqueousmediaareusuallyintheirdissolvedform,whileadifferentsolventoftenrequiressomekindofimmobilization,throughasolidsupportorcrossed‐linkedproteincrystals.Thismakestheprocessmoreadvanced.Itisalsoimportanttoinvestigatetheeffectonwatercontentonenzymebehavior.Toolowamountofwatercommonlyhindersthecatalyticactivity,whereasstabilityisincreased[Halling,2002].

2.6 Gene expression

2.6.1 Recombinant gene expression Toexpressaspecificgene,thegeneisinsertedintoanexpressionvectorandtransformedintoahost.Prokaryoticsystemsareverycommon,butforspecificpurposesandrequirementsfromtheproteinexpressed,eukaryoticsystemssuchasyeast,insectormammaliancellsareavailableaswell.Ofthebacterialexpressionsystems,E.coliisbyfarthemostcommonlyusedforhigh‐levelproteinexpression.Itpresentsanumberofadvantages,includingeasycultivationandmanipulationusingsimpleequipment,availabilityofstrainsandvectorsformaximizedexpression,greatknowledgeaboutthegeneticsandphysiologyofE.coliand,inoptimalcases,rapidproteinproduction[Appelbaum,2003].Although,italsopresentsafewlimitationsforproduction.Theproductcannotbemodifiedaftertranslation,thushinderingprocesseslikeglycosylation,acetylation,amidationandphosporylation[Ling,2001].Morecomplicationswillbefurtherdiscussedindepthlater.

Eachspecificgeneexpressionpresentsitsownchallenges,andageneral,all‐purposeprotocolforgeneexpressioncannotbemade.Staderrecommendsahigh‐copy‐numberexpressionvector,togethigheryields,andatightgeneregulation,becausemanyproteinsaretoxictothecellwhenproducedinhighnumbers[Stader,1995].

2.6.2 Control of gene expression TherelativeamountsofdifferentproteinsinE.colivarybetweenlessthan0.01%uptoabout2%ofthetotal.Thismakesitevidentthatgenetranscriptionisregulatedbythecell.Theseregulationsystemscanalsobeusedforsyntheticproduction.Regulationineukaryotesiscontrolledbyanumberofregulatorysignals,whereasinprokaryotesthemainmethodsaretranscriptionalcontrolandposttranscriptionalcontrol,thelatterconsistingofthedestructionofsynthesizedmRNAandproteinsynthesisrateregulation.Themoreimportantofthetwoistranscriptionalcontrol,consistingofrepressors,operatorsandpromoters[Watson,2007].

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Oneofthemostextensivelystudiedandwell‐usedregulatorsisthelacoperon.Itconsistsofaregulatorygenecodingforarepressorprotein,acontrolsiteconsistingofthepromoterandoperatorandlastlythestructuralgenes,codingforβ‐galactosidase,lactosepermeaseandthiogalactosidasetransacetylase,proteinsthathelpthecellmetabolizelactose.Withoutlactoseavailable,therepressorproteinbindstotheoperatorsite,therebyhinderingtranscriptionofthegene.Whenlactoseisaddedtothecells,itbindstotherepressorprotein,allowingDNApolymerasetobindtothepromoterandtranscribethegenes(Figure2).Innature,thiswouldleadtoamoreefficientlactosemetabolism,butbysubstitutingthetranscriptionalgeneswithageneofinterest,thesamesystemisusedforrecombinantgeneproduction.Lactoseisoftensubstitutedwiththeanalogueisopropylβ‐D‐1‐thiogalactopyranoside(IPTG)[Watson,2007].

Figure2:Theprincipleofthelacoperon,initsrepressedstate.Theregulatorygene(I)producesarepressorprotein(1),whichbinds(2)totheoperator(O),therebyhinderingtranscriptioninitiationatthepromoter(P).Withlactosepresent,step2isobstructedandtherepressorproteindoesnotbindtotheoperator.DNApolymerasethenbindstotheoperatorandisfreetotranscribethestructuralgenes(Z,YandA).

2.6.3 T7 Expression system TheT7expressionsystemisasystemforstrong,regulatableexpressionofaspecificrecombinantgene.ItisbasedontheT7RNApolymerase,isolatedfrombacteriophageT7,capableofelongatingchainsaboutfivetimesfasterthanRNApolymerasefromE.coli.ThebacterialstrainincludesalacUV5promoter,governingtheproductionofT7RNApolymerase.TheadditionofIPTGtothecultureinducesthepromoter,leadingtoproductionofT7RNApolymerase,whichinturnusestheT7promoterintheplasmidtotranscribethetargetDNA[Studier,1990].Thissystemdoesnoteliminatetheneedforrepressionusingarepressorprotein,butchangestherepressingneedsfromtheactualgenetotheT7RNApolymeraseexpressionsite[Stader,1995].

2.6.4 Expression vectors Efficientexpressionvectorscontainastrongpromoterthatcanberegulated,aribosomebindingsequencespecificforE.coliandastrongterminationsequence.Directlyfollowingthetranscriptionstart,amultiplecloningsitecanusuallybefound.Thisisusedtoinsertthegeneofinterest.Becauseofthegapbetweenthetranscriptionstartandthestartoftheactualgene,asmallpeptidefromtheexpressionvectorisfusedtogetherwiththerequestedprotein.Thiscanhaveseveraladvantages.Firstly,thetranslationofmRNAisaffectedbytheribosomebindingsite,butalsothestartofthecodingregionhasbeenshowntointerfere.IntroducingthesequencewithnaturalE.colisequencesisawayofavoidingdifficultieswhenribosomebindingtakesplace.Secondly,thebacterialpeptidemaypreventthepeptideofbeingdegradedbythehostcell,asthecellrecognizesthesequence.Foreignproteinsareoftendestroyedbythehost.Thirdly,theextrasequencecouldbeusedtotagtheproteinwithasignalpeptidethatdirectstheproteintodifferentpositionswithinthecell,intheperiplasmicspacebetweentheinnerandoutermembraneorintotheculturemedium.Theextrasequencecouldalsobe

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optimizedforaffinitychromatographyforeasyrecoveryinthepurificationsteps[Brown,2006].

Inthisstudy,twoexpressionvectorsareexamined(Figure3).pTZ19Risinitiallychosen.Thisvectorhasbeenusedearlierwiththespecificgene.pSE420isusedasanalternativeexpressionvectorandisintroducedinlaterexperiments.

pTZ19Rcontainsageneforβ‐lactamase,givingthehostampicillinresistance,foreasyselectionofcoloniescontainingtheplasmidgrownonplateswithampicillin.Itincludesalacpromoter,andagenecodingfortheN‐terminalfragmentofβ‐galactosidase.Themultiplecloningsiteispositionedbetweenthepromoterandthefragment,allowingforblue/whitescreening.ThevectoralsocontainsaT7promoter,allowingforhighproductionratesofmRNA[Fermentas(2)].

pSE420alsocontainsaβ‐lactamasegeneforampicillinresistance.Itusesthetrcpromoterforhigh‐leveltranscription,thelacOoperonfortranscriptionalregulationusingIPTGandthelacIrepressortoensurethatnoprematureexpressionoccurs[Biocompare].

Figure3:Theexpressionvectorsused:pTZ19RandpSE420withmarkedfeaturesandusedrestrictionsitestogetherwiththeirpositionsinthegene.

2.6.5 Complications with E. coli TherearesomecomplicationsintheexpressionofaforeigngeneinE.coli.Generallyspeaking,theinsertedgenemustnotcontainsequencesthatcanbeinterpretedasterminationsignalsformRNAproduction,thegenemustnotcontainintronsandthecodonbiasofthegenemustbecompatiblewiththatofthehost[Brown,2006].Forexample,only4%oftheleucinsinE.coliareencodedbytheCUAcodonand50%bytheCUGcodon[KazusaDNAResearchInstitute].Ifahighnumberofaminoacidsareencodedbyalesser‐usedcodon,transcriptionmaybehampered.However,theseproblemspredominantlyarisewhileworkingwitheukaryoticgenes,andinthecasethattheyoccurcanbeworkedoutbymodifyingthegene.Otherproblems,withtheirbasisinE.coliitself,arethatposttranslationalmodificationscannotbeperformed;meaning

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proteinsrequiringsuchalterationsneedtobeproducedinanothercelltype.Therecombinantproteinmightnotbefoldedcorrectly,anddisulfidebondsaregenerallynotsynthesized.Errorsinfoldingleadstotheproductionofinclusionbodies,clustersofaggregatedprotein.Lastly,thecellmightdegradetheprotein.Themechanismsbehindthisselectionarelargelyunknown[Brown,2006].

2.6.6 Inclusion bodies Athighexpressionratesofplasmid‐encodedgenes,manypolypeptidesfailtoundergocorrectfoldingandinclusionbodiesconsistingofanaggregatedproteincomplexareformed,oraredegradedbythecell.Toavoidinclusionbodies,differentgrowthparameterscanbetested.Itmightbenecessarytolowertheyieldofproteininordertogetafunctioningprotein[MarCarrió,2001;Georgiou,1996].

Somecommonwaysofloweringtheyieldaretouseaweakpromoterorastrongpromoterunderpartialinductionconditions.Loweringthetemperaturealsodecreasesthedrivingforceforproteinself‐associationandtherateofproteinsynthesis,leadingtolessinclusionbodyformation.Atleastonestudyshowsthatrichmediumcellcultureshelptheproductionofprotein[Georgiou,1996].SomeotherfactorsthatcouldaffectinclusionbodyformationsinvolvesextracellularpH,usedcarbonsource[Strandberg,1991],aminoacidcompositionofthegene,thepresenceofchaperonesandfoldasesandtheexpressionoftheproteinasafusionwithahighlysolublepolypeptide[Georgiou,1996].

Insomecases,inclusionbodiescanprovebeneficial,andbecauseoftheirgeneralstabilitytheycaneasilybeisolatedfromthehost.Itisthenneededtodenaturetheaggregatedproteinandcarefullyallowtherefoldingprocesstotakeplace.However,notallproteinslendthemselvestothiskindofextraction.Also,inlargescalethisisafairlyexpensivetask[Georgiou,1996].

AsolutionofTris,EDTA,lysozymeandTritonX‐100canbeusedtosolubilizethecellenvelopeandreleaseDNA.SonicationreducestheviscositycausedbythedischargedDNAand8Mureaiscommonlyusedforthesolubilizationofinclusionbodies[Stader,1995].

2.6.7 Proteolysis Inanormalcell,theproteolysismachinerymakessurethatmisfoldedproteinsaredegradedanddonotaccumulatewithinthecell.Apartfromcleaningupnon‐functionalpolypeptides,thisallowsforaminoacidrecycling.Targetsforproteindegradationincludeproteinsthathavefoldedincorrectlyandprematurelyterminatedpolypeptides.InE.colicytoplasm,thedegradationisinitiatedbyfiveheatshockproteases,Lon,ClpYQ/HslUV,ClpAP,ClpXpandFtsH.Peptidases,capableofdegradingpeptides2‐5residuesinlengthcontinuethereaction[Baneyx,2004].

2.7 Experimental methods

2.7.1 Restriction endonucleases RestrictionendonucleasesareusedingenecloningtocleaveDNAmoleculesatspecificsites.Theirdiscoveryinthe1960srevolutionizedtheuseofrecombinantDNAtechnology.TherestrictionendonucleasetypeIIrecognizesspecificDNAsequences,frequentlypalindromicsequences,andcleaveatorclosetotherecognitionsite[Mani,2007](Figure4).Itisoftenfavorabletocleavewiththetypeofenzymesthat

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produceasomewhatstaggeredcleavage,resultinginshortoverhangsattheendscalledstickyends[Brown,2006].ThismakesitpossibletoeasierfitdifferentDNAfragmentstogether,inthiscasethegeneexpressingthedehydrogenaseandtheexpressionvector.

Figure4:DNAcleavedbyarestrictionenzyme.XbaIrecognizesthesequenceTCTAGAandleavesthecleavedDNAwithstickyends.

2.7.2 Ligation DNAfragmentsareassembledusingligation.T4DNAligasecatalysestheformationofthebondbetweenthephosphategrouplocatedonthe5’strandandthehydroxylgrouponthe3’strand.Sinceonlyoneofthestrandsneedstobephosphorylatedduringligation,itisusefultotreatthevectorwithadephosphorylasetopreventself‐ligationofvectorsthatarenotfullycleaved.ThedephosporylatedDNAstillcontainsa3’hydroxylgroup,leavingthevectorcapableofligatingwithanon‐treatedDNAfragment[Quail,2005;Ukai,2002].

2.7.3 Polymerase chain reaction Thepolymerasechainreaction(PCR)isanimportanttoolinmolecularbiologytoamplifyDNA.Itisalsoausefulmeantomakemodificationstoplasmids,suchasalteringbases,insertionsanddeletions[Chamberlain,2004].AbriefsummaryoftheprinciplebehindPCRisshowninFigure5.

ThePCRcycleconsistsofthreetemperaturesteps:denaturation,annealingandelongation(Table1).ThedenaturationstepservestoseparatetheDNAstrands,theannealingstepprovidesprimerstickingtothesinglestrandedDNAandduringtheelongationstep,thepolymerasesynthesizesanewcorrespondingstrand.Beforethefirstcycle,thereisalongerdenaturationstepmakingsuretheDNAtemplateiscompletelydenatured,andafterthelastcyclethereisalongerelongationstepmakingsuretheprotrudingendsofthesynthesizedproductiscompletelyfilled.AtypicalPCRconsistsof25‐35cycles[Metzker,2009;Chamberlain,2004].

Table1:RecommendedthermalcyclingconditionsforPfuDNApolymerase[Fermentas(1)]

Step Temperature(°C) Time(min)Initialdenaturation 95 1‐3Denaturation 95 0.5‐2Annealing 37‐70(primerdependent) 0.5‐2Extension 70‐75 2/kbpFinalextension 70‐75 5

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Figure5:TheprincipleofPCR.TemplateDNA(1)isheatedsothestrandsseparatefromeachother(2).Loweringthetemperaturecausesoligonucleotideprimerstoadheretotheloosestrands(3).AheatstabileDNApolymeraseaddstotheprimersequencesandconstructsanewstrandfromthetemplate(4),whichcanbeusedinanewreactioncycle.Figureadaptedfrom[Metzker,2009].

2.7.4 Genetic engineering Geneticengineeringcanbeusedtoalterenzymecharacteristicsor,asinthiscase,correcterrorsinDNAsequencesandmakemodificationstowardsbetterproteinyields.InordertoinsertanucleicacidinanexistingDNAsequence,site‐specificmutagenesisisused.OnemethodtoobtainthisresultisbyusingPCRwithapairofmutagenicprimersthatdoesnotfullymatchthetemplatesequence.Theprimersbindtothetemplateandformthestartingmaterialfornewstrands[Brown,2006].

ThroughPCRandcleverprimerdesignitispossibletoaddarestrictionsitetoanamplifiedgenesequence.Thisisdonebyaddingmismatchingbasesinthe5’endoftheprimer(Figure6)Theremainingbasesoftheprimerbindtothetemplate,andnewlysynthesizedDNAwillcontaintherestrictionsiteadded.ThisisusefulwhenthePCRproductwillbecleavedandputintoanewplasmid.

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Figure6:AddingaEcoRIrestrictionsitetoanewlysynthesizedPCRproductbyaddingthenecessarybasesinthe3’endoftheprimer.Additionalbasescanalsobeadded.

Asequenceofaminoacidscanberemovedfromtheedgesofagenebyintroducingaprimerwithamismatching5’endcontainingarestrictionsiteandastartcodon.Withthestartcodonplacedinsidethegene,theprecedingaminoacidswillnotbeamplifiedandtherestrictionsitecanbeusedtoacquirestickyendsforplasmidinsertion[Chamberlain,2004].

2.8 Kinetic characterization Thekineticpropertiesofanenzymecanbeanalyzedbymeasuringtheinitialvelocityofthereactionandfittingtheresulttoamathematicmodel.ByfarthemostcommonmodelistheMichaelis‐Mentenmodel:

€

V0 =Vmax[S]

[S]+ KM

(1)

Thismodeldescribesthesimplesttypeofenzymaticreactions,andanumberofsimplificationsmustbemadeinordertofitthedatatothemodel.Usinghighstoichiometricamountsofco‐enzymeinrelationtosubstrateconcentrationscanbedonetoobtainapseudo‐first‐orderreaction.Initialvelocityismeasuredasclosetothereactioninitiationaspossibleinordertomakemeasurementsbeforehighconcentrationsofproductareformed[Cornish‐Bowden,2004;Berg,2002].

Graphicalrepresentationscanbeusedtoestimatethekineticconstants.Themostpopularmethodreliesonthedoublereciprocalplot,orLineweaver‐Burkplot(Equation2).Itwasfirstusedinthe1930’s,andeventhoughitcontainsflawssuchasmisleadingimpressionsofexperimentalerroritcontinuepopular.Instead,forshowinghowwellthedataagreestothemathematicalinterpretation,theHanes‐Woolfplotispreferred(Equation3)[Cornish‐Bowden,2004].

€

1V0

=KM

Vmax1[S]

+1

Vmax (2)

€

[S]V0

=1

Vmax[S]+ KM

Vmax (3)

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3 Experimental details

3.1 Cloning

3.1.1 Restriction endonuclease cleaving ThesyntheticgenefordehydrogenasewasdeliveredfromDNA2.0(MenloPark,USA)inacloningvectorwithcleavingsitesandkanamycinresistancegene.Approximately200‐500ngofDNAwasmixedwith1µlofeachrestrictionenzyme,XbaIandSacI(Table2),2µlof10xbufferanddH2Oinatotalreactionvolumeof20µl.Themixturewasincubatedat37°Cfor15minutes.CleavingwasperformedusingFermentasbrandFastDigestenzymestogetherwithbufferprovided.Whencleavedproductwastobeanalyzedonagarosegel,greenbufferfromthesamemanufacturerwasusedinstead,eliminatingtheneedofloadingdye.

Aftercleaving,expressionvectorswerefurtherdephosphorylatedthroughadding1µlofFastAPand2.2µlofFastAP10xbufferandincubatedfor15minutesat37°C.

Table2:Restrictionenzymesused,theirrecognitionsequences,locationsanduses.BoldAmeansthatitrecognizesthemethylatedvariant.

Restrictionenzyme

Recognitionsequence

Locationanduse

Xbal TCTAGA Locatedupstreamfromthegene.UsedwithpTZ19R.SacI GAGCTC Locateddownstream.UsedwithpTZ19RandpSE420.EcoRI GAATTC Locatedupstream.UsedwithpSE420.DpnI GATC About20locations.UsedtodigestmethylatedDNA

3.1.2 DNA separation Agarosegelwasmadebydissolving2gofagarosein200mlofTBEelectrophoresisbufferbyheatinginamicrowaveoven.Aftercoolingforafewminutes,20µlofgelredwasaddedforcolormarkup,gentlymixedandthegelwasmouldedwithasmallorlargecomb,givingthewellsdifferentsizes.Smallwellsholdabout15µlofsampleandlargeholdabout35µl,dependingonhowthegelswerecast.Smallwellsarefirstusedfortestingwithminimalsamplewasteandlargewellsareusedforfurtherpurification.

Productfromrestrictionendonucleasecleavingcanbeloadeddirectly.Smallgelswereloadedbymixing2µlof6xLoadingDyewith10µlofsample,andlargewellswereloadedwith5µlloadingdyeto25µlsample,orproportionalamounts.

AhighandalowMassRulerDNAladderwereloadedoneachgel,andthesampleswereseparatedbyapplying110Vcurrentforabout90minutes.

FinishedgelswerephotographedinUVlightorusedforgelextraction.

AppropriatebandsfromlargewellswereexaminedquicklyinUVlight,cutoutandweighed.GelextractionwasperformedusingQIAquickGelExtractionKitaccordingtomanufacturer’sinstructions,withtheexceptionofelutingin25µlofdH2O.

12

3.1.3 Concentration measurements AllconcentrationmeasurementswerecarriedoutonaCary100BioSpectrophotometer(Varian,PaloAlto,USA),usingquartzcuvettes.Sampleswerediluted1:100indH2O,andabsorbancewasmeasuredat260nmand280nm.ConcentrationwascalculatedusingtheLambert–BeerLaw(

€

A = εlc ),with

€

εdsDNA = 0.020(ng /µl)−1cm−1fordoublestranded

DNA.PurityoftheDNAwithrespecttopolypeptidecontaminationwasmeasuredbyexaminingtheratioA260/A280.

Measuringabsorbancelowerthan0.1isgenerallyconsideredinaccuratebecauseofthehighimpactofnoiseandfluctuations[Sambrook,2001].However,itisconvenienttodoaquicktestwiththesamedilutionfactorforallsamples,bearinginmindtheresultcouldbeimprecise.Onlyasmallamountofsampleisneeded,andtheresultisgenerallytrustable.Fortheseapplicationsitisconsideredsufficient.

3.1.4 Ligation Ligationwasperformedusingdifferentweightratiosofvectorandgenefragment.Theratiosexaminedwere1:1,1:3and3:1.DNAfromvectorandgenefragmentweremixedwith2µlofligasebuffer,1µlofT4DNAligaseand1µlofATP.Thereactionvolumewasadjustedto20µlwithdH2O.Thereactionwasleftat37°Cfortwohours.

3.1.5 Transformation TransformationwasperformedusingfrozencompetentDH5αE.colicellspreparedatthelaboratoryandstoredat‐80°C.Tubeswith100µlDH5αcellswerethawedonice.TheDNAfromligationreactionwasdiluted5‐foldin10mMTris‐HCl(pH7.5)1mMEDTA.PlasmidDNAwasdiluted50‐fold.1µlofDNAwasaddedtothecellsfollowedbyincubationfor30minutesonice.Toperformthetransformation,thecellswereheat‐shockedfor45secondsin42°Cwaterbathbeforebeingputbackonicefortwominutes.900µlofSOCmedium(SeeAppendix)wasaddedtoeachtubeandthecontentswastransferredto15mlplastictubes.Thetubesareincubatedat37°C,180rpmfor1hour.400µlofthecellswerespreadonpreheatedLB/Ampplates(SeeAppendix)andincubatedat37°Covernight.GrownsinglecolonieswerepickedfromtheplateandtransferredontoanewLB/Ampplateandwereincubatedat37°Covernight.Transformedcellswithoriginalgeneconstructwereinsteadgrownonplateswithkanamycin.

3.2 Analytical methods

3.2.1 Plasmid preparation Plasmidpreparationwasusedtoobtainsmallamountsofplasmidsforagarosegelanalysisorsequencing.Abacterialculturewaspickedwithaninoculatorandputin5mlofmedium.LB/Ampmedium(SeeAppendix)wasusedforallapplicationsexcepttheoriginalgeneconstructforwhichLBwithkanamycinwasused.Samplesweregrownovernightat37°C,180rpm.Thesampleswerethenspundownat4000rpmfor15minutesandthesupernatantwasdiscarded.PurificationoftheharvestedcellswascarriedoutusingFermentasGeneJETPlasmidMiniprepKitaccordingtoinstructions,butelutedin30µlofdH2O.Resultingproductwasuseddirectlyorstoredin‐20°C.

3.2.2 Sequencing SequencingwasperformedbyEurofinsMWGOperon(Ebersberg,Germany).Samplesweremadethroughplasmidpreparation,concentrationdeterminedanddilutedindH2Otoholdafinalconcentrationofaround80ng/µlandavolumeof20µl.

13

3.2.3 Cell cultivation Cellcultivationwascarriedoutinautoclavedwide‐neckedErlenmeyerflasks(E‐flasks),withacapofaluminiumfoil.Allhandlingofcellculturestookplaceonasterilebench.

CellsfromafreshplatewasinoculatedintoanE‐flaskwithLB/Ampmediumandgrownovernightunderdifferenttemperatures:25°C,30°Cand37°C.TheculturewasthenscaledupbytransferringintolargerE‐flasksandaddingadditionalLBmedium.ForexactamountsandE‐flasksizes,seeTable3.Afterthreehours,proteinexpressionwasinducedbyaddingIPTGto1µM,oraspecifiedconcentration.After2or4hours,expressionwashaltedbyputtingtheflasksonice.Cellculturewasharvestedbytransferringinto50mlplastictubesandcentrifugedfor15minutes,4000rpm,4°C.Thesupernatantwasdiscarded.

Table3:DifferentculturesizesaswellasE­flasksandmediumvolumesused.

Culturesize

Initialmediumvolume

InitialE­flasksize

Scale­upmediumvolume

Scale­upE­flasksize

Small 5ml 50ml 20ml 100mlMedium 20ml 100ml 80ml 500mlLarge 40ml 500ml 160ml 1000ml

3.2.4 Cell lysis Asmallamountoftheharvestedcellpelletwascollectedusingthetipofapipetteandresuspendedin300µloflysisbuffer(1mg/mllysozymein100mMTrisHCl,pH6.8).Itwasincubatedonheat‐plate,30°C,350rpmfor1hour.Thesamplewasthencentrifugedfor10minutes,13200rpm,andthesupernatantwasrecoveredintofreshtubes.

3.2.5 Protein separation Toanalyzesamplesforproteins,sodiumdocecylsulfatepolyacrylamidegelelectrophoresis(SDS‐PAGE)wasperformed.13µllysedcellsamplewasmixedwith5µlofSDSloadingdyeand2µlofdithiothreiolreducingagent.Themixturewasheatedonheatingplate,70°Cor90°Cfor10minutes.Sampleswereloadedonaprecast10%TEO‐ClSDSgel(CBSScientific,SolanaBeach,USA)togetherwithaproteinstandard.SeeBluePlus2PrestainedStandardwasusedinearlyexperimentsandPagerulerPrestainedProteinLadderwasusedinlaterexperiments.Thegelwasrunat200Vforapproximately45minutes,thensoakedinCoomassieBrilliantBluesolution(2.4%CoomassieBrilliantBlue,60%methanol,12%aceticacidindH2O)foratleast1hour.Gelwasdestainedbysoakingindestainingfluid(10%methanol,10%aceticacidindH2O).

3.2.6 Enzyme activity assays Enzymeactivitywasmeasuredbylettingenzymefromalargeculturereactin50mMglycinebuffer,pH9.5,containing3mMsubstrateand3mMNADP.Thetotalvolumewas10mlinastirredvialthatwaskeptat30°C.

ReductionofNADPwasmeasuredafter24hoursat340nminaspectrophotometer,asdescribedintheliterature[Dell,2009].Measurementsweredoneindisposablepolymethylmethacrylate(PMMA)cuvettes.Samplesfromthereactionwerediluted1:20togiveamaximalabsorbanceof1ifNADPHwasformedto100%,calculatedwithLambert‐BeerLawusingtheextinctioncoefficient

€

εNADP = 6.220mM−1cm−1[Dawson1985].

14

Theenzymeassaywasevaluatedwithastockenzymesolutionofalcoholdehydrogenase(ADH).TwotypesofADHwereusedtogetherwithtwotypesofalcoholsubstrates.TheenzymesusedwereADHTfromThermoanaerobacterandADHTSfromamicrobialsource,bothfromJülichFineChemicals(nowCodexis,RedwoodCity,USA).Substratesusedwere1‐pentanol[II]andα‐phenylethanol[III].UsingLambert‐BeerLawasdescribedbefore,thesampleanalyzedneededtocontain0.16mMNADPinitiallytogiveamaximumabsorbanceof1at100%yield.Amountswereenlarged100timesforthereactionanddilutedformeasurements;thereactionwasthereforerunwithaNADPconcentrationof16mM,using16mM1‐pentanoland32mMα‐phenylethanol.Sincetheenzymesusedarestereospecificandα‐phenylethanolisaracemicsolution,doubleamountswereused.Reactionswereperformedinstirredvialskeptat30°Cwithatotalvolumeof5mlin50mMglycinebuffer,pH9.5.Measurementsweremadeafter24hours.

[II]1­pentanol [III]α­phenylethanol

3.2.7 Inclusion body screening SampleswerescreenedforinclusionbodiesbyadaptingaprotocolfromVanderbiltUniversityforinclusionbodyproteinpurification[PurifyingProteinfromInclusionBodies].

Theharvestedcellpelletfromamediumsizecellculturewasweighedandresuspendedin3volumesoflysisbuffer(50mMTris‐HCl,pH8.0,5mMEDTA,10mMNaCl).PMSF(100mMin99.5%ethanol)wasaddedtogiveafinalconcentrationof1mM,and16µloflysozyme(50mg/mlin100mMTris‐HCl,pH6.8)wasaddedforeachgramofcellpellet.Thetubewasmixedandputat37°Cinawaterbathfor20minutes.Theresultingviscoussolutionwastransferredto25mlbeakersandsonicatedat20%amplificationfor30secondswithaVibracellVCX750sonicator(Sonics&Materials,Newtown,USA)usingaCV33probe.Thesolutionwasthendividedintosmallertubesandcentrifugedfor30minutesat13200rpm.Thesupernatantwasremoved,butsavedforfurtheranalysis.Thecellpelletisresuspendedinthesameamountofbuffer(20mMNa2HPO4,pH7.2,20mMNaCl,5mMEDTA,25%(w/v)sucrose),PMSFwasaddedtogiveafinalconcentrationof1mMand10µlofTritonX‐100wasaddedpermlsolution.Thesolutionwascentrifugedfor30minutesat13200rpm.Supernatantwasremoved,butsavedforfurtheranalysis.Thecellpelletwasresuspendedin8.0Mureasolutionina50°Cwaterbath.

3.3 DNA modifications

3.3.1 Implemented modifications ThreeDNAmodificationswereperformed,resultingineightclones.ThepTZ19RexpressionvectorwaschangedtopSE420toperformsimultaneousexperimentswithanalternativevector.Toavoidinclusionbodyformationpossiblycausedbyextraaminoacidsaddedtotheenzymebyexpressionvectordesign,thegenewasshortenedwith12

15

or13aminoacidsforthepTZ19RvectorandpSE420vector,respectively.Finally,sequencingofthecodinggenerevealedanerrorconsistingofamissingguanidine,whichwasadded.PCR‐basedmethodswereusedtomodifytheDNA.Thefirsttwoalterationswereperformedinparallelbyamplifyingfragments,andthelastbyamplifyingthewholeplasmid.

3.3.2 Polymerase chain reaction PCRwasperformedinaUnoCyclerThermalCycler(VWRInternational)capableofrunningagradientoftemperaturesinasinglerun.OligonucleotideprimersweredeliveredfromEurofinsMWGOperon(Ebersberg,Germany).AlistofprimermeltingtemperaturescanbeseeninTable4.

PCRsamplemixturewaspreparedbymixingthefollowinginthin‐walledPCRtubes:39.5µlofdH2O,5µlof10XPfuBufferwithMgSO4,1µlofDNAtemplate(1ng/µl),1µlofdNTPmix(10mMofeachbase),1µlofforwardprimer(20µM),1µlofreverseprimer(20µM)and1.5µlofPfupolymerase(1.5u/µl),resultinginatotalreactionvolumeof50µl.

PCRoffragmentswasperformedusingaprogramconsistingof60secondsinitialdenaturationat95°C,followedby30cyclesof30secondsdenaturationat95°C,45secondsannealingat55°C,60°Cand62°Cand90secondselongationat72°Candendedwithanadditionalelongationstepfor5minutesbeforecoolingto4°C.

Whole‐plasmidPCRwasperformedwith60secondsofinitialdenaturationat95°C,followedby18cyclesof30secondsdenaturationat95°C,60secondsannealingat53°C,58°Cand60°Cand12minuteselongationat72°C.Theprogramwasendedwith12minutesofadditionalelongationbeforecoolingto4°C.

Table4:PrimersusedinfragmentPCR(firstgroup)andwhole­plasmidPCR(secondgroup)withtheirrespectivemeltingtemperaturesascalculatedbythemanufacturer.

Primername Meltingtemperature

Use

Xbalshortforward 64.6°C Introductionofrestrictionsiteandshorteningofgene

EcoRIshortforward 64.8°C Introductionofrestrictionsiteandshorteningofgene

EcoRIlongforward 65.0°C IntroductionofrestrictionsiteSacIreverse 65.1°C Correspondingrestrictionsiteforallclones Ginsertforward 63.2°C InsertionofguanineataspecificlocationGinsertreverse 63.0°C Insertionofguanineataspecificlocation

3.3.3 Cloning of modified DNA CloneswithmodifiedDNAwereobtainedusingmethodsdescribedearlier:5µlofPCRfragmentswereseparatedonagarosegeltoevaluatepurityandsuccessrate.Theremainderofsuccessfulsamplesweretreatedwithrestrictionenzymesandseparatedonagarosegel.Concurrently,pTZ19RplasmidstreatedwithXbalandSacIandpSE420plasmidstreatedwithEcoRIandSacI(Table2)wasalsoseparatedonagarosegel.BandswithsampleandplasmidswerecutoutandpurifiedusingQIAquickGelExtractionKit

16

andconcentrationwasmeasured.Plasmidsandfragmentswereligatedovernightat37°C.Ligationproductswereusedfortransformation.

Whole‐plasmidPCRproductswereevaluatedbyseparating5µlonagarosegel,andsuccessfulsamplesweretreatedwithDpnIrestrictionenzymetodegradetheoriginaltemplate.TheproductwaspurifiedandconcentratedusingQIAquickPCRPurificationKit,poolingthesamplesofthesamekindandelutingin10µlofdH2O.Theresultingproductwasuseddirectlyfortransformationwithoutfurtherdilution.

3.4 Kinetic characterization AnassayforkineticcharacterizationofdehydrogenasewasmadeandtestedwithADHTfromThermoanaerobacter,usingthesameprinciplesasfortheactivitytests.ReactionswerecarriedoutinPMMAcuvettesandmeasuredspectophotometrically.Reactioncomponentswereaddedtoobtainafinalvolumeof1ml.Astheenzymewasadded,thecuvettewasquicklymixedbyinvertingandinitialreactionvelocitywasmeasuredduringthefirst10secondsofthereaction.Enzymeamountwaskeptconstantat0.15UandNADPat5mM.Thesubstrateconcentrationwerevariedbetween0.005and1.0mMineightmeasurementpoints(0.005,0.01,0.025,0.05,0.1,0.25,0.5,1.0mM).Reactionswerecarriedoutinglycinebuffer(50mMglycine,pH9.5)atroomtemperature.

17

4 Results and discussion

4.1 Protein expression ThegenefordehydrogenasewasclonedfromthevectoritwasdeliveredinandinsertedintothepTZ19Rexpressionvector.TheresultingplasmidwastransformedintoDH5αE.coliandgrownonLB/Ampplates.Transformedcellsgrewwell.Theresultwasanalyzedonagarosegel,asseeninFigure7.BoththepTZ19Rvectorandtheoriginalsyntheticgenevectorhaveapproximatelythesamemolecularweight(b).Thegenefragment(a)ispresentinallsamplesbuttheemptypTZ19Rvector(well2),asexpected.Thismeansthatthecloningstepshavesucceededandgeneexpressioncanbestarted.

Figure7:Agarosegelseparationofgeneandvector.1:Originalgeneconstruct,2:EmptypTZ19Rvector,3,4,5:DehydrogenasegeneinsertinpTZ19R,a:Genefragment,b:Vector

Todetermineanidealgrowthandexpressiontemperature,cultureswiththedehydrogenasegeneweregrownat37°C,30°Cand25°C.Acombinedtestwheregrowthtookplaceat37°Candexpressionat30°Cwasalsoperformed.Thecellswereinducedwithafinalconcentrationof1.0mMIPTGandharvestedaftertwohoursofexpression.AculturewithemptypTZ19Rvectorswasusedasnegativecontrol.SamplesweretakenforSDS‐PAGEseparation(Figure8).Thegelseparationdidnotrevealanyclearbandspresentinanyoftheculturesgrowndifferingfromthenegativecontrol,nordidthedifferenceindenaturingtemperatureseemtohaveaneffect.However,thegelrevealsdifferencesinintensityofthebands,wherehighercultivationtemperaturesappeartogiverisetoastrongerbackgroundofcellproteins.

Activitytestingwasinitiatedtoexamineiftherewouldbeanysignsofactiveenzymepresentinthesamples.Twolargebatchesofenzymewerepreparedfortwotemperatureconditions:25°Cgrowthandexpressionand37°Cgrowth,30°Cexpression.Allavailableenzymefromlysedcellswasusedintheassay.However,noenzymaticactivitycouldbedetected.HPLCmeasurementswereperformedtoconfirmthatthesubstratehadnotspontaneouslydecomposed.

18

Figure8:InitialSDS­PAGEseparationofcellculturesgrownandexpressedat25°C,30°C,37°Candasamplegrownat37°Candexpressedat30°C.Twodifferentdenaturingtemperatures(inparenthesis)areused.1:37/30°Csample(70°C),2:37°Csample(70°C),3:Negativecontrol(70°C),4:30°Csample(70°C),5:25°Csample(70°C),6:37/30°Csample(90°C),7:37°Csample(90°C),8:Negativecontrol(90°C),9:30°Csample(90°C),10:25°Csample(90°C).

ToconfirmthattheenzymaticassaywasfunctionalandthatNADPwasactive,anenzymeassayusingstockalcoholdehydrogenaseandalcoholswasprepared.Amountswerechosenastocorrespondto0%yieldatabsorbance0at340nm,and100%yieldatabsorbance1,givinganapproximationoftheactivity.Table5showsthatADHTexhibits16%yieldandADHTS52%yieldwith1‐pentanol,whilebothenzymesshow100%yieldwhenα‐phenylethanolisusedasasubstrate.Theseresultsalsosuggestthatmeasurementsusingthisassayweresomewhatinaccuratesincethemeasuredabsorbancesurpasses1.0.

Table5:Absorbancemeasuredinactivitytestingoftwotypesofalcoholdehydrogenaseandtwotypesofsubstrates.Negativecontroldoesnotcontainanyenzyme.

Substrate Enzyme Absorbance340nm1‐pentanol ‐ 0.01521‐pentanol ADHT 0.15791‐pentanol ADHTS 0.5232α‐phenylethanol ‐ 0.0212α‐phenylethanol ADHT 1.0532α‐phenylethanol ADHTS 1.0642

ToinvestigatetheeffectofdifferentIPTGconcentrations,cellsweregrownat30°CandinducedwithsixdifferentIPTGconcentrations:0.01mM,0.1mM,0.25mM,0.5mM,0.75mMand1.0mM.SampleswereanalyzedonSDS‐PAGE.Nosignificantvariationinexpressioncouldbedetected.

Inconclusion,activeenzymecouldbefoundneitheratSDS‐PAGEanalysis,noratenzymeactivityassays.Thereasonbehindthismightbeformationofinclusionbodiesduringexpression.Apossibleexplanationforinclusionbodyformation,evenatlowertemperaturesandIPTGconcentrations,mightbethetwelveextraaminoacidsthatwereattachedtotheproteinfromtheexpressionvector.Thissequencecouldcausetheproteintofoldincorrectly,thusrenderinginutileprotein.Itwasalsoconcludedthatusingabetter‐knownexpressionvectorcouldhelpunderstandingtheproteinexpression.Threenewclonesweredesigned,onewithashortergeneinthepTZ19R

19

vector,andtwousingthenormalgeneandashortervariantinthepSE420vector.ThecloneswerepreparedbyfragmentPCRusingappropriateprimersandthreedifferentannealingtemperatures,seeFigure9.Allsamplesexceptsample7wereconsideredfunctionalforfurtherligationandtransformation.

Figure9:AgarosegelseparationofPCRfragmentsfortheconstructionofcloneswithshortergeneandpSE420expressionvector.Wellcontents:SamplestreatedwithXbalshortforwardprimerandannealingtemperaturesetto55°C(1),60°C(2)and62°C(3).SamplestreatedwithEcoRIshortforwardprimerandannealingtemperaturesettoto55°C(4),60°C(5)and62°C(6).SamplestreatedwithEcoRIlongforwardprimerandannealingtemperaturesetto55°C(7),60°C(8)and62°C(9).

Uponcompletion,transformedsamplesweresentforsequencingandexpressionwasstudiedonthenewclonesusingthesamemethodasbefore.Testswerecarriedoutin37°C(Figure10)and30°Cusing1mMIPTGand2hourexpressiontime.Theresultswerenegative;nogeneexpressioncouldbeidentifiedonthegels.Furthertestingwasabortedduetotheresultsofthesequencing.

Figure10:SDS­PAGEseparationofcellculturesgrownat37°C.1:Negativecontrol,2:OriginalgeneinpTZ19R,3:ShortenedgeneinpTZ19R,4:OriginalgeneinpSE420,5:ShortenedgeneinpSE420.

SequencingoftheclonesshowedthattheDNAmodificationshadbeenexecutedcorrectly.However,adeletionofasingleguaninenucleotidewasfoundneartheendofthegenesequence,confirmedinallfivesequencesthatcoveredthisregion.Thisindicatedthattherewasanerrorinthegenesequencepresentinalloftheclones,thusprobablyoriginatingfromanerrorintheoriginalgene.Thisframeshiftofthetranslationofthefollowingnucleotidesleadstomisreadingofthestopcodonandtheproteinexpressioncontinuesaftertheproteinshouldhaveended.Thiscouldbethereasonbehindthelackofgeneproductintheanalysis.

Therefore,inordertocorrectthemissingnucleotide,theexpressionvectorswerecopiedusingPCRwithprimersdesignedwiththemissingbaseinserted.Theoriginal

20

templatewasdigestedwithDpnIendonucleasestoeliminateplasmidswithouttheinsert.AscanbeseeninFigure11,copynumberswerelow.

Figure11:AgarosegelseparationofwholeplasmidPCRproductswithinsertedguanine.Wellcontents:pTZ19RGinsertwithannealingtemperatureof53°C(1),58°C(2)and60°C(3).pTZ19RshortGinsertwithannealingtemperatureof53°C(4),58°C(5)and60°C(6).pSE420Ginsertwithannealingtemperatureof53°C(7),58°C(8)and60°C(9).pSE420shortGinsertwithannealingtemperatureof53°C(10),58°C(11)and60°C(12).

Simultaneously,amethodforexaminingthecellpelletforinclusionbodieswasinvestigated.SamplesweretakenfromdiscardedsupernatantsduringtheprocessandseparatedonSDS‐PAGEgels.Expressionwasmeasuredonthenewcloneswithinsertedguanineusingtheinclusionbodyscreeningprotocolinordertoexamineboththesolubleandinsolubleproteincontent.Expressiontimewasincreasedtofourhourstoinvestigateifalongerexpressiontimecouldhelpaugmenttheyield.SDS‐PAGEgelsshowednonewbandsineitheranalysis.

Figure12:SDS­PAGEseparationofinclusionbodyscreeningsamplesgrownat30°Ccontaining:firstcentrifugationsupernatantofnegativecontrol(1)andexpressionsample(2),secondcentrifugationsupernatantofnegativecontrol(3)andexpressionsample(4),8MUreasuspensionofnegativecontrol(5)andexpressionsample(6).

21

Insummary,Table6givesanoverviewofperformedanalysisofproteinexpressionbasedonthedehydrogenasegene.

Table6:Asummaryofgeneexpressionanalysisofdehydrogenaseperformedwithdifferentgenemodifications,underdifferentgrowthtemperaturesandexpressiontimes.

Genemodification Temperature Time AnalysispTZ19R 25°C 2h SDS‐PAGE,Enzymaticactivity 30°C 2h SDS‐PAGE,Inclusionbodyscreening,

IPTGgradient 37°C 2h SDS‐PAGE 30/37°C 2h SDS‐PAGE,EnzymaticactivitypTZ19RGinsert 25°C 4h SDS‐PAGE,Inclusionbodyscreening 30°C 2h SDS‐PAGE,Inclusionbodyscreening 37°C 4h SDS‐PAGE,InclusionbodyscreeningpTZ19Rshort 30°C 2h SDS‐PAGE 37°C 2h SDS‐PAGEpTZ19RshortGinsert 25°C 4h Inclusionbodyscreening 37°C 4h InclusionbodyscreeningpSE420 30°C 2h SDS‐PAGE 37°C 2h SDS‐PAGEpSE420Ginsert 25°C 4h Inclusionbodyscreening 37°C 4h InclusionbodyscreeningpSE420short 30°C 2h SDS‐PAGE 37°C 2h SDS‐PAGEpSE420shortGinsert 25°C 4h Inclusionbodyscreening 37°C 4h Inclusionbodyscreening

4.2 Kinetic characterization ThemethodsforcharacterizationweretestedwithADHfromThermoanaerobacterusingα‐phenylethanolasasubstrate,toprepareforkineticcharacterizationofthesoughtdehydrogenase.Thechosenamountsofinvolvedchemicalswerefoundempirically,withrespecttakentohaveasteady,nearlinear,reactionduringthefirstminute.

TheMichaelisMentenplot(Figure13)comparesthesubstrateconcentrationwithinitialvelocity.Overtime,thegraphconvergestowardsthemaximumvelocity(Vmax)ofthereactionwiththisenzymeconcentration.Reviewingequation1showsthatKMisthesubstrateconcentrationathalfofthisvalue.ThekineticconstantscanbeextractedusingtheLineweaver‐Burkgraphicalrepresentation,wherebothaxesareinverted(Figure14).Inthisgraph,theyinterceptequals1/Vmaxandthexinterceptequals‐1/Km.FromthisaVmaxvalueof90.9µmol/minutewascalculatedandthecorrespondingKmwas164µM.

Vmaxisthevalueofmaximumenzymaticactivity.ComparingwithFigure13,thetruevaluesoftheconstantsareprobablylowerthancalculated.ThevaluescanalsobecalculatedfromtheHanes‐Woolfplot(Figure15),whereVmaxwascalculatedto66.7µmol/minuteandthecorrespondingKMto113µM.OnceagaincomparingwithFigure13,thesevaluesseemmoreplausible.

22

Figure13:KineticmeasurementsofalcoholdehydrogenasefromThermaanaerobacter,followingtheMichealis­Mentenmodel.Theinitialreactionvelocity(v0)isplottedagainstthesubstrateconcentration([S])inthreeseries,withaveragevaluesplottedasaline.

Figure14:KineticmeasurementsofalcoholdehydrogenasefromThermaanaerobacter,inaLineweaver­Burkplot.Thereciprocalinitialreactionvelocity(1/v0)isplottedagainstthereciprocalsubstrateconcentration(1/[S])inthreeseries.Thelinerepresentslinearregressionofaveragevalues,withtheequationinserted.

Figure15:KineticmeasurementsofalcoholdehydrogenasefromThermaanaerobacter,inaHanes­Woolfplot.Substrateconcentrationisdividedwiththeinitialreactionvelocity([S]/v0)andplottedagainstthesubstrateconcentration([S])inthreeseries.Thelinerepresentslinearregressionofaveragevalues,withtheequationinserted.

010203040506070

0 100 200 300 400 500 600 700 800 900 1000Initialvelocity[µmol/m

in]

Substrateconcentration[µM]

y=1,804x+0,011

0

0,1

0,2

0,3

0,4

0,5

0 0,05 0,1 0,15 0,2

1/Initialvelocity

[min/µmol]

1/Substrateconcentration[µM­1]

y=0,015x+1,698

0

5

10

15

20

0 100 200 300 400 500 600 700 800 900 1000Substrateconcentration/

Initialvelocity[min/l]

Substrateconcentration[µM]

23

Inordertocomparethisenzymetoothers,theamountofenzymeusedisanimportantfactortoconsider.Sinceenzymeconcentrationisunknowninthestocksolution,absorbanceat280nmismeasuredtoestimatethisvalue.Theconcentrationismeasuredata1:100dilution,andgaveanabsorbanceof0.4274.

Usingtheestimatedextinctioncoefficient

€

εADH = 30.940mM−1cm−1[SwissInstituteof

Bioinformatics]andassuming100%purity,thestockenzymeconcentrationisestimatedto1.4mM,or52grams/liter.Furthermore,thissignifiesthateachreactioncontained2.6µgenzyme.Thus,thecalculatedspecificactivityofthisenzymeis26000µmolmin‐1mg‐1forreactionswithα‐phenylethanolasasubstrate.Thisvalueisanestimate;bearinginmindthatreactionconditionssuchastemperatureisnotoptimalandtheconcentrationcalculatedisapproximate.

24

5 Conclusions ThedehydrogenasegenewassuccessfullyclonedintoDH5αE.colicellsusingthepTZ19Rexpressionvectoraspreviouslydescribedintheliterature.However,initialanalysisofproteinexpressionindicatedthattheenzymewasnotexpressedcorrectly.Themostprobableexplanationwasformationofinclusionbodiesandtodealwiththis,threenewclonesweresetuptobeanalyzedsimultaneously.Toinvestigatetheimpactoftheexpressionvector,thegenewasclonedintothepSE420vectorasanalternative.

Sincethecombinationofrestrictionsitesandexpressionvectorscaused12extraaminoacidstobeaddedtothebeginningofthegene,thiswasthoughtasapossiblecauseforinclusionbodyformation.Therefore,12aminoacidswereremovedfromthebeginningofthegene,substitutedwiththeaminoacidscodedbytheexpressionvector.TheusageofarestrictionsitecontainingtheATGstartsequence,suchasNcoI(CCATGG)orNdeI(CATATG)wouldhaveminimizedthislossifavailableandcorrectlysituatedinthereadingframe.However,availablerestrictionsitesintheexpressionvectorswerealsopresentinthegene,therebyblockingtheirusage.

SequencingofthefinishedDNAmaterialrevealedanerrorinthegene,asinglenucleotidedeletion,presentinallfourclones.Sincethiscausesaframeshiftofthetranslationofthegene,renderingallfollowingaminoacidschanged,thegeneticcodewascorrectedinallclones.Followingtestingofgeneexpressionstillindicatedthattheproteinwasnotexpressedcorrectly.Analysisofinclusionbodycontentinexpressedcellsalsoshowedlittleornoexpressionoftheprotein.

Additionally,theoriginalgenesequenceusedwasfoundtocontainerrorscausedbyusinganobsoleteversionofthesequencereleasedbytheauthors.Theerrorconsistsofasingleaminoacidchangeinonelocationandasequenceofsixaminoacidschangedinanotherlocation.Thiserrorcouldrenderanon‐functionalprotein,butshouldhowevernotimpactproteinexpression.

Thereareafewpossibleexplanationsastowhytheproteinisnotexpressedcorrectly.SincetheproteinisnotnativetoE.coli,thecodonusageintheproteinmightbesuboptimalforexpressionoutsideitsnativehost,therebyhamperingproteinexpression.Anotherpossibleexplanationisthattheproteinisdegradedbythecell.TheDH5αstrainofE.colicontainsgenesforLonprotease,naturallyusedbythecelltodegrademisfoldedorunfoldedproteins.Iftheproteinisfoldedincorrectly,itmightbedegradedbytheseproteases,therebynotshowingupinanalysis.

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6 Recommendations Correctingtheerrorsremaininginthegeneticsequencemightberequiredfortheexpressionofactiveprotein.Thereisachancethattheintroducederrorsdonotaffectproteinfolding,buttherewilllikelybeapositiveeffectinthefinishedproductiftheseaminoacidsarecorrect.ItispossibletousemultiplePCR:stocorrecttheseinaccuracies.

Thecodonusageinthegeneshouldbeoptimized,usingthemostcommoncodonsinthehostforanefficienttranslationprocess.However,doingthisusingPCRwillproveatediousprocesswhereonlyafewbasescanbeaffectedforeachcyclerun.Acompletelynewgene,usingtheupdatedsequenceandoptimizedcodonsmightbeabetteralternative,albeitcostly.

Toimpedeproteolyticactivity,abacterialstrainknockedoutforproteasesshouldbeused.TheBL21strainofE.coli,whichcanbeusedlicence‐free,fallsunderthiscategory.BL21(DE3),cellslysogenizedwiththeDE3phage,arealsocapableofusingtheT7expressionsystemavailableonthepTZ19Rexpressionvector.TheT7systemisoftenusedforhighexpressionratesandmightproveanadvantage.

Afterproteinexpressionisobtained,itshouldbeoptimizedwithrespecttotemperature,IPTGconcentration,thetimeofinductionandexpressionduration.Growthconditions,suchasmediumusedtogetherwiththesubstrate,shouldbedetermined.Theactivitytestproposedinthisreportcanbeusedtoassesstheefficiencyoftheenzyme.

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7 Acknowledgements IwouldliketothankCambrexKarlskogaABgivingmetheopportunitytodothisthesisandthestaffforwelcomingmeintotheworkplace.SpecialthanksgoestomysupervisorCeciliaBrannebyforinvaluablehelpinthelaboratory,discussionsandwhilewritingthisthesis,LinaLindbergforhelpingmeinthelaboratoryandforendlessdiscussions,ThomasNorström,forHPLCanalysisandPärHolmbergfordiscussionsaroundthesubstrate.

IwouldalsoliketothankmyexaminerUnoCarlssonandLars‐GöranMårtenssonatLinköpingUniversityforinterestinginputonmyresults,andmyopponentAnnelieBorgström.

Finally,IwouldliketothankChristinaandAndersRubensonforwelcomingmetoKarlskogaandkindlyofferinghousingduringmystay,familyandfriendsforyourencouragement.

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8 References AppelbaumER,ShatzmanAR:Procaryoticinvivoexpressionsystems,InSJHiggins,BDHames(Eds):ProteinExpression:aPracticalApproach,Oxford:OxfordUniversityPress,2003

BaneyxF,MujacicM:RecombinantproteinfoldingandmisfoldinginEscherichiacoli,NatureBiotechnology,2004;22(11):1399‐1408

BergJM,TymoczkoJL,StryerL:Biochemistry,5thed.,NewYork:W.H.FreemanandCompany,2002

Biocompare:pSE420ExpressionvectorfromSigma­Aldrich,Availableonlineat:<http://www.biocompare.com/ProductDetails/349908/pSE420‐Expression‐vector.html>(Downloaded2010‐02‐15)

BothamKM,MayesPA:BiologicOxidation,InMurrayRK,BenderDA,BothamKM,KennelyPJ,RodwellVW,WeilPA(Eds):Harper'sIllustratedBiochemistry,28thed.,NewYork:McGraw‐Hill,2009

BrownTA:GeneCloningandDNAanalysis,5thed.,Oxford:BlackwellPublishing,2006

BuggTDH:IntroductiontoEnzymeandCoenzymeChemistry,2nded.Oxford:BlackwellPublishing,2004

CarrióMM,VillaverdeA:Proteinaggregationasbacterialinclusionbodiesisreversible,FEBSLetters,2001;489(1):29‐33

ChamberlainJ:PCR­mediatedmutagenesis,InEncyclopediaofLifeSciences,Chichester:JohnWiley&Sons,<http://www.els.net>,2004

Cornish‐BowdenA:FundamentalsofEnzymeKinetics,3rded.,London:PortlandPress,2004

DawsonRB:Dataforbiochemicalresearch,3rded.,Oxford:ClarendonPress,1985

DellEJ,GanskeF:DetectionofNADHandNADPHwiththeOmega’sHighSpeed,FullUV/VisAbsorbanceSpectrometer,BMGLabtechInc.Availableonlineat:<http://www.analytica‐world.com/articles/e/86152>(Downloaded2009‐12‐03)

FaberK:BiotransformationsinOrganicChemistry:ATextbook,4thed.,Heidelberg:SpringerVerlag,2000

Fermentas:(1)ProtocolforPCRwithPfuDNAPolymerase,productinformationforPfuDNAPolymerase

Fermentas:(2)pTZ19RDNA.Availableonlineat:<http://fermentas.com/en/products/all/molecular‐cloning/vectors‐phage/sd014‐ptz19r‐dna>(Downloaded2010‐02‐15)

GeorgiouG,ValaxP:ExpressionofcorrectlyfoldedproteinsinEscherichiacoli,CurrentOpinioninBiotechnology,1996;7(2):190‐197

HallingP:EnzymicConversionsinOrganicandOtherLow­WaterMedia,InDrauzK,WaldmannH(Eds.):EnzymeCatalysisinOrganicSynthesis,Weinheim:Wiley‐VCH2002

28

HummelW,KulaMR:Dehydrogenasesforthesynthesisofchiralcompounds,EuropeanJournalofBiochemistry,1989;184(1):1‐13

IshigeT,HondaK,ShimizuS:Wholeorganismbiocatalysis,CurrentOpinioninChemicalBiology,2005;9(2):174‐180.

KazusaDNAResearchInstitute:CodonUsageDatabase,Entry:EscherichiacoliW3110.Availableonlineat:<http://www.kazusa.or.jp/codon/cgi‐bin/showcodon.cgi?species=316407>(Downloaded2010‐03‐31)

LingMM:GeneExpressioninEscherichiacoli,InEncyclopediaofLifeSciences,Chichester:JohnWiley&Sons,<http://www.els.net>,2001

LiuW,WangP:Cofactorregenerationforsustainableenzymaticbiosynthesis,BiotechnologyAdvances,2007;25(4):369‐384

ManiM,KandavelouK,ChandrasegaranS:RestrictionEnzymes,InEncyclopediaofLifeSciences,Chichester:JohnWiley&Sons,<http://www.els.net>,2007

MetzkerML,Caskey,CT:PolymeraseChainReaction(PCR),InEncyclopediaofLifeSciences,Chichester:JohnWiley&Sons,<http://www.els.net>,2009

PankeS,HeldM,WubboltsM:Trendsandinnovationsinindustrialbiocatalysisfortheproductionoffinechemicals,CurrentOpinioninBiotechnology,2004;15(4):272‐279

PurifyingProteinfromInclusionBodies,Availableonlineat<http://structbio.vanderbilt.edu/chazin/wisdom/labpro/inclusion.html>(Downloaded2009‐12‐12)

QuailMA:DNACloning,InEncyclopediaofLifeSciences,Chichester:JohnWiley&Sons,<http://www.els.net>,2005

SambrookJ,RusselDW:Molecularcloning,alaboratorymanual,3rded.ColdSpringHarbor:ColdSpringHarborLaboratoryPress,2001

SchoemakerHE,MinkD,WubboltsMG:DispellingtheMyths–BiocatalysisinIndustrialSynthesis,Science,2003;299(5613):1694‐1697

StaderJ:GeneExpressioninRecombinantEscherichiacoli,InSmithA(Ed):GeneExpressioninRecombinantMicroorganisms,NewYork:MarcelDekker,1995

StudierFW,RosenbergAH,DunnJJ,DubendorffJW:UseofT7RNAPolymerasetoDirectExpressionofClonedGenes,MethodsinEnzymology,1990;185:60‐89

SwissInstituteofBioinformatics:ExpasyProtParam,Entry:SwissProtaccessionnumberP14941,Avalableonlineat<http://expasy.org/tools/protparam.html>

UkaiH,Ukai‐TadenumaM,OgiuT,TsujiH:Anewtechniquetopreventself‐ligationofDNA,JournalofBiotechnology,2002;97(3):233‐242

WatsonJD,CaudyAA,MyersRM,WitkowskiJA:RecombinantDNA,3rded.,NewYork:W.H.FreemanandCompany,2007

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Appendix: Growth medium Thegrowthmediumusedwasastandardlysogenybroth(LB)consistingof:

• 10goftryptone• 5gofyeastextract• 5gofNaCl• dH2Otoatotalvolumeof1literandautoclavedat121°Cfor20minutes.

Toobtainaselectivemedium,ampicillinwasaddedtogiveafinalconcentrationof100µg/ml.

Agarplatesweremadeinthesamefashionwiththeadditionof15gofagarose.

KanamycinplateswerepreparedbyusingLBplatesandadding30µlofkanamycinin100µlofdH2O.Thismethodwasfunctionalandeliminatedtheneedofmakingkanamycin‐specificplatesastheywereusedinlownumbers.

Intransformation,anextrarichmediumwasused.TheSuperOptimalBroth(SOB)basewasmadebeforehandandtheunstablefinishedcataboliterepressionmedium(SOC)waspreparedrightbeforeuse.

SOB:(1liter)

• 20goftryptone• 5gofyeastextract• 0.58gofNaCl• 0.19gofKCl• 4mlofNaOH,1M• dH2Otoatotalvolumeof1literandautoclavedat121°Cfor20minutes.

SOC:(1ml)

• 960µlofSOB• 10µlofMgCl2• 10µlofMgSO4• 20µlofglucose,1M,sterilized