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Purification and Characterization of C. elegans Mitochondrial Malate Dehydrogenase

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004232/00001

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Title: Purification and Characterization of C. elegans Mitochondrial Malate Dehydrogenase
Physical Description: Book
Language: English
Creator: Cassidy, Emma
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2010
Publication Date: 2010

Subjects

Subjects / Keywords: C. elegans
Malate dehydrogenase
Proteins
Biochemistry
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: C. elegans Malate Dehydrogenase (MDH-1) was isolated and purified from E. coli cells using an Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system (NEB). C. elegans is a small nematode worm which has long been considered an ideal model organism for scientific research due to it�s heartiness, transparency and relative simplicity. Malate dehydrogenase (MDH), which catalyzes the reversible conversion of malate into oxaloacetate, is a protein common to almost all living cells. It has been studied quite extensively due to its important role in several metabolic pathways, most notably the citric acid cycle. MDH has been characterized in many organisms, including bacteria and mammalian models; however, there has been very little research done on the C. elegans mitochondrial isoform, MDH-1. Better understanding of the structure and function of this enzyme may offer insight into the enzyme�s activities in higher organisms, which are more difficult to study. The C. elegans mdh-1 gene was ligated into a the plasmid vector pTXB1, adjacent to an intein sequence and a chitin binding domain (CBD), and transformed into ER2566 E. coli cells. The plasmid and cell design allowed for IPTG-induced expression of the target gene. The expressed MDH-1 was fused to the intein- CBD tag at its C-terminus. The intein is a peptide which cleaves at its N-terminus upon exposure to DTT. Cell lysate from induced cultures were applied to a chitin bead column, which bound the MDH-1-intein-CBD. DTT was then applied to the column to cleave the intein tags, and the MDH-1 was eluted from the column. The MDH-1 samples were sub jected to enzyme assays. at room temperature. The Km for oxaloacetate was 26.6 ?M, and the specific activity was 4.6 Units/mg.
Statement of Responsibility: by Emma Cassidy
Thesis: Thesis (B.A.) -- New College of Florida, 2010
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2010 C34
System ID: NCFE004232:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004232/00001

Material Information

Title: Purification and Characterization of C. elegans Mitochondrial Malate Dehydrogenase
Physical Description: Book
Language: English
Creator: Cassidy, Emma
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2010
Publication Date: 2010

Subjects

Subjects / Keywords: C. elegans
Malate dehydrogenase
Proteins
Biochemistry
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: C. elegans Malate Dehydrogenase (MDH-1) was isolated and purified from E. coli cells using an Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system (NEB). C. elegans is a small nematode worm which has long been considered an ideal model organism for scientific research due to it�s heartiness, transparency and relative simplicity. Malate dehydrogenase (MDH), which catalyzes the reversible conversion of malate into oxaloacetate, is a protein common to almost all living cells. It has been studied quite extensively due to its important role in several metabolic pathways, most notably the citric acid cycle. MDH has been characterized in many organisms, including bacteria and mammalian models; however, there has been very little research done on the C. elegans mitochondrial isoform, MDH-1. Better understanding of the structure and function of this enzyme may offer insight into the enzyme�s activities in higher organisms, which are more difficult to study. The C. elegans mdh-1 gene was ligated into a the plasmid vector pTXB1, adjacent to an intein sequence and a chitin binding domain (CBD), and transformed into ER2566 E. coli cells. The plasmid and cell design allowed for IPTG-induced expression of the target gene. The expressed MDH-1 was fused to the intein- CBD tag at its C-terminus. The intein is a peptide which cleaves at its N-terminus upon exposure to DTT. Cell lysate from induced cultures were applied to a chitin bead column, which bound the MDH-1-intein-CBD. DTT was then applied to the column to cleave the intein tags, and the MDH-1 was eluted from the column. The MDH-1 samples were sub jected to enzyme assays. at room temperature. The Km for oxaloacetate was 26.6 ?M, and the specific activity was 4.6 Units/mg.
Statement of Responsibility: by Emma Cassidy
Thesis: Thesis (B.A.) -- New College of Florida, 2010
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2010 C34
System ID: NCFE004232:00001


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PuricationandCharacterizationof C.elegans MitochondrialMalateDehydrogenase By EmmaCassidy AThesis SubmittedtotheDivisionofNaturalSciences NewCollegeofFlorida Inpartialfulllmentoftherequirementsforthedegree BachelorofArts UnderthesponsorshipofDr.KatherineWalstrom Sarasota,FL May,2010

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Thankyou!

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Contents Introduction 1 Malatedehydrogenase....................................1 MDHPartiticipatesinSeveralCellularPathways.....................5 C:elegans asaModelOrganism..............................10 CloningwiththeIMPACTsystem.............................12 Materials&Methods15 LigationofMDH-1intopTXB1andtransformationinto E:coli cells..........15 InducingTargetGeneExpression..............................18 Site-directedMutagenesisofInteintag/MDH-1interface.................22 ComparingMutated,Non-mutatedandNo-insertplasmids...............28 Enzymeassays........................................30 DialysisandProteinConcentrationAssay.........................31 Results&Discussion32 Summary...........................................32 LigationofMDH-1intopTXB1andtransformationinto E:coli cells..........32 InducingTargetGeneExpression..............................33 Site-DirectedMutagenesis..................................34 ComparingMutated,Non-mutatedandNo-insertplasmids...............37 FinalPuricationonChitinColumn............................40 EnzymeCharacterization..................................45 Conclusions 49 SolutionsAppendix51 References 54 iii

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ListofFigures 1ComparisonofMDHandLDHSubstrates......................1 2ReactionmechanismofMalateDehydrogenase...................3 3OxidizedandReducedNicotinamide.........................5 4TricarboxylicAcidCycle................................6 5ReactionSchemeforSubstrateChannelingStudies.................7 6GlyoxylateCycle....................................8 7Malate-AspartateShuttle...............................10 8IMPACTSystemSchematic..............................13 9Thiol-inducedInteinCleavageMechanism......................14 10pTXB1VectorPolylinker...............................16 11SiteDirectedMutagenesisSchematic.........................23 12SiteDirectedMutagenesis,ShortGene........................24 13SiteDirectedMutagenesis,LongGene........................24 14ScreeningPlasmidsfor mdh-1 Insert.........................33 15ScreeningPlasmidsafterSite-directedmutagenesis.................35 16ScreeningPlasmidsafterSite-directedmutagenesisControl...........35 17Small-scaleinductionofMutatedL410Eclones...................36 18Chitincolumnpurication,MutatedLclone....................37 19Small-scaleInductions,allMutatedL4ClonesWesternblot...........38 20SequencealignmentfororiginalpTXB1with mdh-1 longinsert,L4,L410C...39 21L410CChitinColumnPuricationSamplesWestern...............41 22L410CElutionFractionsGel............................42 23L4notmutatedChitinColumnPuricationSamplesWestern.........43 24L4notmutatedElutionfractionsGel.......................44 25Michaelis-MentenCurve,OAAconcentrationvs.V 0 ................46 26ProteinConcentrationStandardCurve........................47 27QIAprepSpinMiniprepKitProcedure........................53 iv

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ListofTables 1LigationReactionmixtures..............................16 2DoubleDigestReactionmixtures...........................17 3SiteDirectedMutagenesisReactions.........................26 4DoubleDigestReactionmixtures...........................27 5ProteinConcentrationofL410CE3ElutionFractions...............46 6ComparisonofK m andSpecicActivityValuesformMDHinVariousOrganisms48 v

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PuricationandCharacterizationof C.elegans MitochondrialMalateDehydrogenase EmmaCassidy NewCollegeofFlorida,2010 ABSTRACT C.elegans MalateDehydrogenaseMDH-1wasisolatedandpuriedfrom E.coli cellsusingan InteinMediatedPuricationwithanAnityChitin-bindingTagIMPACTsystemNEB. C. elegans isasmallnematodewormwhichhaslongbeenconsideredanidealmodelorganismfor scienticresearchduetoit'sheartiness,transparencyandrelativesimplicity.MalatedehydrogenaseMDH,whichcatalyzesthereversibleconversionofmalateintooxaloacetate,isaprotein commontoalmostalllivingcells.Ithasbeenstudiedquiteextensivelyduetoitsimportantrole inseveralmetabolicpathways,mostnotablythecitricacidcycle.MDHhasbeencharacterized inmanyorganisms,includingbacteriaandmammalianmodels;however,therehasbeenvery littleresearchdoneonthe C.elegans mitochondrialisoform,MDH-1.Betterunderstandingof thestructureandfunctionofthisenzymemayoerinsightintotheenzyme'sactivitiesinhigher organisms,whicharemorediculttostudy. The C.elegansmdh-1 genewasligatedintoatheplasmidvectorpTXB1,adjacenttoanintein sequenceandachitinbindingdomainCBD,andtransformedintoER2566 E.coli cells.The plasmidandcelldesignallowedforIPTG-inducedexpressionofthetargetgene.Theexpressed MDH-1wasfusedtotheintein-CBDtagatitsC-terminus.Theinteinisapeptidewhichcleaves atitsN-terminusuponexposuretoDTT.Celllysatefrominducedcultureswereappliedtoa chitinbeadcolumn,whichboundtheMDH-1-intein-CBD.DTTwasthenappliedtothecolumn tocleavetheinteintags,andtheMDH-1waselutedfromthecolumn.TheMDH-1samples weresubjectedtoenzymeassays.atroomtemperature.TheK m foroxaloacetatewas26.6 M ,andthespecicactivitywas4.6Units/mg. Dr.KatherineWalstrom DivisionofNaturalSciences vi

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Introduction Malatedehydrogenase MalatedehydrogenasesaremembersofalargeanddiversefamilyofNAD + nicotinamide adeninedinucleotide{dependentdehydrogenases.TheenzymesuseNAD + asacoenzymeinthe catalysisofthefollowingreversiblereaction: Malate + NAD + Oxaloacetate + NADH + H + ThisreactionreplacesahydroxygroupwithacarbonylgroupattheC-2positionofthesubstrate molecule.Itisanalogoustothepyruvate-lactatereactionthatLactateDehydrogenaseLDH performs,andtheiractivesitesaresimilarasaresultBirkoft1983.Manyparallelsaredrawn betweenthecatalyticmechanismsofthesetwoenzymes.AcomparisonofMDHandLDH substratescanbeseeninFigure1. Figure1:ComparisonofMDHandLDHSubstrates. Bothenzymeshave2-hydroxy-aciddehydrogenaseactivity. Inmostorganisms,MDHsarehomodimerswithsubunitsrangingfrom30-35kDa.LDHs aregenerallyhomotetramericMinariketal.2002.HomotetramericMDHsexistaswell,but areuncommon.ThereisnoevidencetosuggestthatMDHsubunitsparticipateincooperative bindingorcatalysisGoward&Nichols1994. 1

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DiversityofMDHIsoforms Inmammalianandyeastcells,twoisoformsofMDHaregenerallypresent;mitochondrial mMDH,whichoperatesintheCitricAcidcycle,andcytosoliccMDHsometimescalledsoluble, sMDH,whichparticipatesinthemalate/aspartateshuttle.Bacteriatraditionallyhaveonly oneMDHisoform,whichinsomecaseshavehighersequenceidentitywitheukaryoticmMDHs andinothercasesresembleeukaryoticcMDHs.Interestingly,however,agivenmMDHexhibits generallyhighersequenceidentitytoaprokaryoticMDHthantoacMDH.Thisobservation suggeststhatmMDHdivergedmorerecentlyfromprokaryoticMDHthanfromcMDH,which alignswiththetheorythatmitochondriawereonceprokaryoticendosymbiontsMinariket al.2002.CertainarchaealMDHshavehighsequenceidentitytothesimilarenzymelactate dehydrogenaseLDH,indicatingacloseevolutionarytiebetweenthesetwoenzymesGoward &Nichols1994. PlantcellsexpressspecializedMDHisoformsintheirchloroplastsandglyoxysomes.ChloroplasticMDHhasaNADP + -dependentmechanismandparticipatesinamalate-oxaloacetate shuttlebetweenthechloroplastandthecytosol.Malate-aspartateshuttlesallowcellstotransportreducingagentsacrossselectivelypermeablemembranes,aswillbediscussedlater.ChloroplasticMDHcanalsohelpfacilitateC 4 carbonxationGietl1992. GlyoxysomalMDHparticipatesintheglyoxylatecycle,whichallowsplantsandsomeother organismstoconvertfatsintocarbohydrates.ThiscycleisdiagrammedinFigure6. Allplantisoformsareexpressedwithamino-terminalextensions,whichactaslocalization signals.ThesetagsarecleavedfromtheMDHisoformsuponreachingtheirrespectivecellular destinations,beitthemitochondria,glyoxysomeorchloroplastGietl1992. Ingeneral,three-dimensionalstructureisstrikinglysimilarbetweendierentisoformsof MDH,despitelowsequenceidentity.Hall&Banaszakfoundthatthecrystalstructures of E.coli MDHandporcinecMDHwereessentiallyidentical",despiteonly20%sequence identity.Asmightbeexpected,thefewabsolutelyconservedresiduesthatexistarestructurally andcatalyticallycrucialones,suchasactivesiteresiduesandthoseinvolvedincoenzymebinding orthesubunitinterface. ActiveSiteandSubstrateSpecicity ThereactioncatalyzedbyeveryMDHisoformisthereversibleconversionbetweenmalate andoxaloacetate.TheactivesiteofMDHisahydrophobiccavity,enclosedbyamobileloop structurethatcontainsimportantresiduesforcatalysisFigure2. Threeargininesintheactivesitepocketareessentialforsubstratespecicityandcatalysis. Twooftheseargininescoordinatewiththecarboxylgroupsoneitherendofthesubstrateshown 2

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Figure2:ReactionmechanismofMalateDehydrogenase,withrelevantresiduesforbindingand catalysis. A:OxaloacetatemoleculeB:NADHReduced.Modiedgure,Goward&Nichols 1994 inFigure2asArg171andArg102Goward&Nichols1994.ThethirdarginineArg109in Figure2stabilizesthehydroxyl/ketogroupwhichformsduringcatalysisBell2001.Twoof thethreesignicantargininesArg102andArg109arelocatedonthemobileloopwhichcloses overtheactivesiteuponsubstratebinding.Withthemobileloopintheclosedposition,bulk solventisexcludedfromtheactivesite. ThesubstratespecicityofbothMDHandLDHhasbeenthesubjectofmuchresearch.In theclosedloopformofMDH,Arg102coordinateswiththeC-4carboxylgroupofthesubstrate whileArg109stabilizestheC-2hydroxy/ketogroupFigure2.Ithasbeensuggestedthat,in theabsenceofthenegativelychargedC-4carboxylgroup,thesetwopositivelychargedsidechains wouldexperiencesignicantrepulsion.ThedicarboxylicacidsubstrateisfavoredsinceitneutralizessomeofthepositivechargeandallowsforthelooptocloseovertheactivesiteBell 2001.InLDH,thenaturalsubstrateisamonocarboxylicacidpyruvate,andresidue102is conservedasaneutralGlutamine.Duetothesendings,residue102hasbeenthetargetfor 3

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manysite-directedmutagenesisstudiesintheseenzymesGoward&Nichols1994. MechanismofCatalysis ThecoenzymeNADHbindsintheactivesiteoftheMDHrst,thenthesubstrateBirkoft 1983.ThereactioniscatalyzedbyapairedHis-Aspprotonrelaysystem,acommoncomponent in2-hydroxyaciddehydrogenasesBell2001,Birkoft1983.Birkoftwasthersttodescribethis pairinMDH.ThehydrogenbondingbetweentheHisandAspallowtheHistoactasboth anacidandbasewithintheenzymeactivesiteFigure2,His195andAsp168.Hisactsas ahydrogenbonddonortotheketo-oxygenoftheoxaloacetatemoleculeintheactivesite,as showninFigure2.ThesameHisactsasanH-bondacceptorwhenmalateisboundinthe activesiteBirkoft1983.AHis/Asprelaysystemrequiresveryprecisealignmentofthe2hydroxylsubstrate.IthasbeensuggestedthatArg171playsasignicantroleinthisalignment Bell2001.TheNADHcoenzymefacilitatestheredoxreactionbyactingasareducingagent, transferringahydridefromtheC-4ofthenicotinamideringtotheC-2oftheoxaloacetate.The entirestructureofNADHanditsorientationintheMDHactivesitecanbeseeninFigure2.A comparisonoftheoxidizedandreducedformsofthenicotinamideringareshowninFigure3. CoenzymeSpecicity NADHbindinginvolvessignicanthydrogenbondingwithatomsintheproteinbackbone, inadditiontoseveralsidechaininteractions.Thereisoftenlowsequenceidentitybetweenthe NADHbindingsitesindierentNADH-dependentenzymes,butveryhighstructuralsimilarity Hall&Banaszak,1993.Keysidechain-NADHinteractionsarewellconservedbetweenMDH andLDHisoformsBirkoft1983. Thesidechainofresidue53showninFigure2asAsp53isconsistentlyacidicacrossMDH isoforms,usuallypresentingasaGluorAsp.ThisresiduebindstheNADHmoleculebyforming hydrogenbondswiththetwooxygensontheadenineribosering.Italsohelpstodetermine coenzymespecicitybyrepellingthenegativephosphategroupofNADPHTomita2006.The NADP + -dependentchroroplastMDHsofsorghumandmaizehaveaglycineatresidue53instead Goward&Nichols1994,ostensiblytoleaveroomfortheadditionalphosphategroupofthe NADP + 4

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Figure3:ComparisonofOxidizedandReducedNicotinamideRings MDHPartiticipatesinSeveralCellularPathways TricarboxylicAcidCycle ThetricarboxylicacidcycleTCA,alsoknownastheKrebscycleortheCitricAcidcycle,is asetofreactionsassociatedwithaerobicrespiration,andistherebypartoftheoverallmetabolic processofturningmacromoleculesintoenergyforthecell.Thecycleoccursinthemitochondrial matrix.PyruvatemoleculesproducedinglycolysismustbeconvertedtoacetylCoAcatalyzed bypyruvatedehydrogenasebeforethecyclecanbegin.TheacetylCoAiscombinedwith anoxaloacetateOAAmoleculetoformcitrate,whichistherstproductofthecycle.Six moreenzyme-mediatedreactionsfollow,convertingthecitratetoisocitrate, -ketoglutarate, succinate,succinyl-CoA,fumarate,andmalate.Malatedehydrogenasethencatalyzesthenal step,convertingmalateintooxaloacetateandcompletingthecycle.Theentirecycle,including allintermediatesandcatalyticenzymes,isshowninFigure4.NADHandFADHareproduced atseveralstepswithinthecycle,includingthemalatedehydrogenationstep.Thesereducing agentsaresenttotheelectrontransportchain,wheretheyareoxidizedinordertoproducea protongradientthatfuelsATPproduction. Theconversionofmalatetooxaloacetateisveryenergeticallyunfavorable,witha G 'of +28.6kJ/mol.Thereactionispushedforwardbycitratesynthaseusingtheoxaloacetateto restarttheTCA,andNADHbeingoxidizedbyComplexIoftheElectronTransportChain.By havingtheproductsproceedimmediatelytosubsequentreactions,theunfavorablereactionis eectivelycoupledwithafavorableoneMolenaar,2000. Enzyme-EnzymeInteractionsandSubstrateChanneling Strategiesforcouplingenzymaticreactionsarenotuncommonincells.Enzymesinvolved withsequentialstepsinspecicpathwaysofteninteractwithoneanother,sometimesforming largecomplexescalledmetabolons.IntheTCA,forexample,sixoftheeightenzymesinvolved inthecycleinteractdirectlywithoneanother.Oftentheseinteractionsincludedirecttransferof 5

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Figure4:TricarboxylicAcidCycle. Garrett&Grisham1999 aproducttotheactivesiteofthefollowingenzyme,astrategytermedsubstratechanneling." Channelingisbenecialtometabolicpathwaysbecauseitincreasestheowofsubstratesthrough thepathwaysandpreventsthelossofintermediatesintosolution,wheretheybecomeavailable tootherenzymes/pathwaysMorgunov&Srere1998. InstudiesofsubstratechannelinginvolvingMDH,itwasfoundbyfrontalanalysisgelltrationchromatographythatsignicantandspeciccomplexingoccursbetweenmMDHand CitrateSynthaseCSbutnotbetweencMDHandCS.ThistechniquebasicallyinvolvedrunningtheMDHthroughacolumnwith10%polyethyleneglycolPEGandmeasuringthe elutionvolumewithandwithoutCSinthecolumn.PEGisusedasacrowdingmolecule,to simulatecellularconditionsandstabilizetheproteincomplexesinsolution,theyquicklydissociate.InthecaseofmMDH,theelutionvolumeoftheproteindecreasedsignicantlywhenCS waspresent,duetotheformationofmMDH-CScomplexes.InthecaseofcMDH,theelution volumewasunchangedinthepresenceofCSMorgunov&Srere,1998.ThelackofcMDH-CS 6

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complexingexempliesthefunctionalspecicitybetweenMDHisoformsandindicatesthatthe mMDHistheonlyisoformactiveintheTCA. TheexperimentalsomeasuredtheconversionofmalatetocitrateviaoxaloacetatebyMDHCScomplexes,precipitatedinPEG,inthepresenceofotherenzymesthat`trap'intermediate OAA.ThetwotrappingenzymesusedwereaspartateaminotransferaseAAT,whichconverts theOAAtoaspartate,andoxaloacetatedecarboxylaseOAADC,whichconvertstheOAAinto pyruvate.ThecompetitionforOAAasasubstratecanbeseeninthereactionschemeinFigure 5.Itwasfoundthat,insolutionswithAATingreatexcess,themMDH-CScomplexmaintained 66%ofit'sactivity.InthecaseofOAADC,themMDH-CScomplexmaintained78%ofit's activity.Thus,neithercompetingenzymewasabletoeectivelytraptheOAAintermediate, duetoprotectedtransferofOAAbetweentheMDHandCS.Whentheseassayswereperformed withoutPEG,themMDH-CScomplexlikelyactingasfreeenzymesinsolutionmaintainedonly 26-28%oftheiractivity,indicatingsuccessfultrappingoftheintermediatesubstrate.Whenthe sameassayswereperformedusingcMDH,thecMDH-CS`complex'whenprecipitatedinPEG maintainedthesameactivityasthefreeenzymesinsolution,againindicatingnointeraction betweentheenzymes.Morgunov&Srere1998. Figure5:ReactionSchemeforSubstrateChannelingStudies. OAADecarboxylaseandAspartateAminotransferasewereusedaspotentialtrapsforintermediateOAA,inordertoobserve substratechannelingwithintheMalateDehydrogenase-CitrateSynthasecomplex.Morgunov& Srere,1998 SimilarcomplexingandchannelingmayoccurbetweenMDHandComplexIalsoknownas NADHDehydrogenase/NDH/NADH:ubiquinoneoxidoreductaseoftheelectrontransport chain.Thischannelingwasobservedin E.coli usingcompetitionassays. E.coli hastwoNDH enzymes;NDH-1alsoComplexIandNDH-2asingle-subunitNDH,notassociatedwiththe 7

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electrontransportchain.IncompetitionassayswithMDH,NDH-1andNDH-2insolution,it wasshownthattotalNDHactivitywasseverelyinhibitedwhenComplexIwasdeactivated.This suggeststhattheNDH-2hadnoaccesstotheNADHproducedbytheMDH.Inthesamestudy, overallNDHactivitywasunaectedwhentheComplexI-specicsubstratedeamino-NADHwas used.ThisindicatesthatComplexIwashasdirectchanneledaccesstotheNADHproduced bytheMDHreactionAmarneh&Vik2005. Glyoxylatecycle Bacteria,protists,plants,fungi,andnematodesusetheglyoxylatecycletoconvertacetylCoAfromfattyacidsintosugars.Thiscyclerequirestwospecialenzymes,isocitratelyaseICL andmalatesynthaseMS.Nematodes,including C.elegans, haveanovelbifunctionalenzyme whichhasbothICLandMSactivity.ItisthoughttohaveevolvedduetothefusionoftheICL andMSgenesKondrashovetal.2006.Theglyoxylatecycleoccursinthecytoplasm. Figure6:TheGlyoxylateCycleallowsforcertainorganismstoproducesuccinatefromacetyl Co-Aunits. C.elegans andothernematodeshaveonebifunctionalenzymewhichperformsboth theisocitratelyaseandmalatesynthasereactionsKondrashovetal.2006.FigurefromGarrett &Grisham,1999. Thecycle,showninFigure6,beginsbyfollowingtherstthreestepsoftheTCAcycle.ICL thensplitsisocitrateintoglyoxylateandsuccinate,andthesuccinateproducedisusedprimarily forgluconeogenesis.MSfusesanotheracetyl-CoAtotheglyoxylatemolecule,formingmalate, 8

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whichre-entersthecycle.Theglyoxylatecyclethusconservescarbonunitsbybypassingthe twooxidativedecarboxylationreactionsintheTCAwhichreleaseCO 2 Malate-AspartateShuttle TheinnermembraneofthemitochondriaispermeableonlytoH 2 O,CO 2 andO 2 .Thisdesign isnecessaryformaintainingtheprotongradientthatdrivesATPsynthesis,andforlocalizing andcompartmentalizingmetabolicpathways.However,Complextransportsystemsareneeded tomovemoleculesacrossthemembrane.TheMalate-aspartateshuttleisawayforcellstomove NADHfromthecytosolintothemitochondria,inordertosupplementtheNADHproducedby theTCAcyclewhichisoxidizedintoNAD + byComplexI. TheshuttleutilizesboththemitochondrialandcytosolicversionsofMDH,andcreatesa nettransferofreducingagentsintothemitochondrialmatrixFigure7.Intherststep, malatemovesintothemitochondrialmatrixinexchangeforan -ketoglutarate,viathemalate-ketoglutaratetransporterintheinnermitochondrialmembrane.MalateisconvertedtooxaloacetatebycMDH,producingoneNADH.Theoxaloacetateisthenconvertedtoaspartate viaglutamate-oxaloacetatetransaminaseGOT,alsoaspartateaminotransferase.Meanwhile, inthecytosol,GOTconvertsthe -ketoglutarateintoglutamate.Glutamatethenentersthe matrixinexchangefortheaspartate,viatheGlutamate-Aspartatetransporter.Onceinsidethe miochondria,glutamateisconvertedbackto -ketoglutarate.Theaspartateinthecytosolis convertedtooxaloacetate,whichisconvertedtomalatebymMDH,producingoneNAD + Gluconeogenesis Asimilarstrategy,utilizingbothmMDHandcMDH,isemployedfortransportingOAA acrosstheinnermitochondrialmembraneforgluconeogenesis.Pyruvatecarboxylase,amitochondrialenzyme,convertspyruvatetooxaloacetate.Thenextstepofgluconeogenesisoccurs inthecytoplasm,sotheoxaloacetatemustbeconvertedtomalate,broughtacrossthemitochondrialmembrane,thenreconvertedtooxaloacetate.Onceinthecytosol,theoxaloacetateis convertedtophosphoenolpyruvatebytheenzymephosphoenolpyruvatecarboxykinase,andthe pathwaycontinues.ThistransportalsobringsreducingequivalentsNADHintothecytosol, whicharethenusedinsubsequentreactionsduringgluconeogenesisBerg2007. 9

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Figure7:Malate-AspartateShuttleSchematic.Twocyclesarehappeninginoppositedirections: 1theconversionof -ketoglutaratetoglutamateinthecytosolandviceversainthemitochondrialmatrixand2Theconversionofmalatetoaspartateandviceversa,viaoxaloacetate Garrett&Grisham1999. C:elegans asaModelOrganism Caeorhabditiselegansisafree-living,transparentnematodewhichisabout1mmlongas anadult.Thewormsliveinsoilandfeedonbacteriaandfungi.Theyhave6pairsofchromosomes-vepairsofautosomesandthesexchromosome,X.Mostwormsarehermaphroditic, withanXXgenotype.Malesexistaswellandarehemizygous,withonlyoneXchromosome. Thehermaphroditesundergoself-fertilizationandeachwillproduceabout300progeny.After hatchingfromanegg,thewormsproceedthroughfourlarvalstagesbeforereachingadulthood, andliveforabouttwoweeks.Insituationswithscarceresources,thewormsmayalsoenteran alternativedauerlarvalstagewheretheycanmove,butdonoteatorage.Theycanexistas suchforuptoseveralmonths,ideallyuntillocatingmoreabundantresources.Thewormsthen entertheL4stageandproceedtoadulthoodwithnoeectonadultlifespan.Metabolicactivity duringthisdauerstagehasbeenthesubjectofextensivestudyHolt&Riddle2003. C.elegans isaextremelyimportantorganismtothestudyofbiology,anditisusedwidelyas amodelorganism.Thegenome,only9.7X10 7 basepairslong,wasthersteukaryoticgenome 10

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tobesequencedinitsentirety.Thewormsaremulticellular,butalsoextremelysimple.Thus, thespeciessharesthetraitsofhigherorganismsforexample,complexdevelopmentalpathways butonsuchasmallscalethatitcanbestudiedingreatdetail.Theinformationgleanedcan thenbeappliedtohigherorganismsthataremoredicultmorallyorlogisticallytostudy -likehumans.Theworm'stransparencyalsoallowsforexaminationofcellularprocessesand dierentiationwithinthelivingorganism. C.elegans arequiteheartyandeasytorearinalaboratoryenvironment.Theyremainviable afterbeingfrozenandthawed,andcanbestoredforlongperiodsoftime.Theshortlifecycle andquickgenerationalturnovermakeslaboratoryinvestigationconvenient. C:elegans MDH-1 The mdh-1 genewormbase:F20H11.3encodestheenzymeMDH-1,amitochondrialmalate dehydrogenaseNCBI:NP498457.ThisgenehashighsequencesimilaritytoseveralmitochondrialandglyoxysomalMDH's.MDH-1operateswithintheTCA,andmayalsoparticipatein theglyoxylatecycleduringembryogenesis.Anothergenewormbase:F46E10.10hasbeendescribedwhichmightencodeacytosolicMDHforthisspecies.TheassociatedproteinNCBI: NP504656hasbeenlabeledMDHCinseveralpublications.Ithasstrongsequencesimilarity withhumancytosolicMDH1,butwaspreviouslylistedasapossibleLDH.Thisproteinislikely toparticipateinoxaloacetateformationforgluconeogenesisHolt&Riddle2003. Researchon C.elegans MDH-1isverylimited.PreviousNewCollegestudentshaveattemptedpuricationandcharacterizationwithmixedsuccess.Inpreviousprojects,MDH-1was expressedwithpolyhistidinetagsattheN-terminusandpuriedonnickelcolumns.Inthis scheme,thecelllysateisrunthroughanickelcolumn,andthehistidinetagsbindtothenickel. Theremainingcellcomponentswerewashedaway,andtheproteinwaselutedfromthenickel resinusingimidazole.Thistechniquealonedidnotproduceverypuresamples,whichhada negativeeectonthespecicactivityBondi2002.Mostrecently,anadditionalpurication stepwasaddedtotheprocedureinanattempttoreducethenumberofcontaminatingproteins. Beforethenickelanitycolumn,thecelllysatewasrunthroughaDEAEion-exchangesepharose column.Inthiscolumn,MDH-1boundtheDEAEresinatpH9andelutedatpH8.Onlythe mostconcentratedsamplesfromtheDEAEcolumnwerepuriedonthenickelcolumn.This double-puricationproducedconfusingresultsduetotheco-puricationof E.coli MDHand C. elegans MDH-1ontheDEAEcolumn.Thenalsamples,however,weresignicantlypurerthan thoseobtainedbyanitychromatographyaloneShanks2008. 11

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CloningwiththeIMPACTsystem Inthisproject,theInteinMediatedPuricationwithanAnityChitin-bindingTagIMPACTsystemwasusedtopurify C.elegans malatedehydrogenaseMDH-1.Figure8shows aschematicoftheIMPACTpuricationprocess.ThesystemusesthevectorpTXB1NEB #N6707,whichincludesaninteintag,about28kDa,witha6kDachitinbindingdomain. Inteinsarepeptideswithinducibleself-cleavingactivity.Theinteinincludedinthisvectorwas amodiedversionof198-aminoacidresidueinteinfromthe Mycobacteriumxenopi gyrAgene MxeGyrAintein. Thetargetgene mdh-1 wasligatedintothevectorsothat,uponexpression,theMDH-1 C-terminuswasadjacenttotheinteintag.Thevectorwastransformedinto E.coli cellsand overexpressedasaMDH-1-Intein-CBDprecursor.TheCBDontheinteintagallowedforanity bindingonachitincolumn.Then,uponexposuretothethiolreagentDTT,theinteinunderwent self-cleavageatitsN-terminus,thusreleasingthepuriednativetargetprotein.Figure9shows themechanismoftheInteincleavage. 12

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Figure8: InteinMediatedPuricationwithanAnityChitin-bindingTagIMPACTSystem Schematic.A:Targetgene C.elegansmdh-1 isligatedintothepTXB1vector.B:ThepTXB1mdh-1 istransformedintoER2566 E.coli cellsandexpressionoftheMDH-1-intein-CBDprecursorisinduced. C:Inducedcellsarelysedandloadedontoachitincolumn.ThecolumnbindstheCBDonthetag,and othercellcomponentsarewashedaway.D:CleavageoftheinteintagisinducedwithDTT.seeFigure 9E:Nativetargetproteinelutedanddialyzed. NEB 13

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Figure9:Thiol-inducedInteinCleavageMechanismFigure8,D.Thetargetproteinisexpressedwithanintein-CBDtag,andboundtoachitincolumn.Theinteincleaveswhenexposed toDTT,allowingforelutionofthenativetargetprotein.Step1:AnN-SShiftoccurs,replacing thepeptidebondwithaC-Sbond.Step2:TheDTTDithiothreitol,HS-CH 2 -CHOH 2 -CH 2 SHattacksattheC-Sbond NEB. 14

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Materials&Methods TheInteinMediatedPuricationwithanAnityChitin-bindingTagIMPACTsystemwas usedtopurifytheenzyme C.elegans malatedehydrogenaseMDH-1.Thetargetgene mdh-1 wasligatedintothevectorpTXB1NEB#N6707,whichwastransformedinto E.coli cells. Whenexpressed,theproteinMDH-1wasfusedtoaninteintagcontainingachitinbinding domainCBDatitsC-terminus,whichfacilitatedpurication.Assayswereconductedto determinethespecicactivityofthepuriedMDH-1. LigationofMDH-1intopTXB1andtransformationinto E:coli cells Thetargetgene, C.elegans malatedehydrogenase mdh-1 ,wasampliedviaPCRand puriedbyapreviousstudent.Twoprimers,oneshortandonelong,wereusedtoamplifythe geneandthePCRproductslabeledLandSaccordingly.ThesePCRproductsweredigested withNde1andXho1andquantitatedonanagarosegel.ThevectorpTXB1NEB#N6707 waspuriedfrombacteriaanddigestedwiththesamerestrictionenzymes.Thelocationofthe restrictionsitesinthevectorpolylinkercanbeseeninFigure10.TheLandSPCRproducts, withstickyends,werebothligatedtothepTXB1vectorinabouta7:1ligationusingT4DNA ligase.Becausetheinsertandtheplasmidhadthesamestickyends,theyboundreadily.T4 DNAligasethensealedtheholesinthephosphatebackbone.Anotherligationreactionwasrun withnoinsertasacontrol.ThereactionsolutionrecipesareinTable1.Eachligationreaction wasrstrunat20 Cfor20minutesandthen65 Cfor10minutestodenaturetheligase.After severalunsuccessfulligationattempts,thiswasalteredto16 Cfor2hoursandthen65 Cfor 10minutes,andanewermoreactiveT4DNAligasewasused. ThepTXB1-mdh-1ligationsweretransformedinto E.coli cells.Separatetubeswith25 L of E.coli cellseachreceived1 LofaligationreactionS,L,orcontrol.Duetopipetting issues,about10 LoftheLandcontrolmixtureswerelost.Thecellswereheat-shockedina42 Cwaterbathfor30seconds,thenplacedin0.5mLicecoldSOCmediaandrecoveredat37 C 15

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Figure10:pTXB1VectorPolylinker. Highlighted:XhoI,XbaI,yellowandNhe1blue. The mdh-1 genewasligatedintothevectorusingrestrictionsitesNde1andXho1.Later,adoubledigestofplasmidprepsampleswasconductedusingXhoIandXbaI. NEB Table1:LigationReactions. mdh-1wasligatedtothepTXB1vectorusingT4DNAligase Ligation: Control S L pTXBI ng=L 1 L 1 L 1 L Linsert ng=L 1 L Sinsert ng=L 1 L T4DNALigase 1 L 1 L 1 L LigaseBuerseebelow 2 L 2 L 2 L NPH 2 0 16 L 15 L 15 L intheshakerat200rpmfor1hour.TheSandLtransformationswerespreadonLB-amp-agar plateswithasterilizedglassrod,in25 Land75 Laliquots.A75 Lcontrolplatewasspread aswell.SincethepTXB1plasmidimpartsampicillinresistance,onlysuccessfullytransformed cellswereabletogrowontheLB-amp-agar.Theplateswereinvertedandincubatedovernight at37 C.Inthemorning,eachSandLplatecontainedabout20-60colonies.Thecontrolplate showednogrowth.Tenculturetubes,eachwith5mLLB-amp,werepreparedandlabeled S1-S5,L1-L5.OneSorLcolonywascarefullytransferredtoeachtubeusingaame-sterilized wireloop.Colonieswereculturedovernightat37 C.Thecellswerecentrifugedfor2minutes at13,000rpminto1.5mLcentrifugetubesandstoredat-20 C. Screeningforplasmidinserts Plasmidpreparationswereperformedonall10cellsamples,usingthestandardprotocolfrom theQIAprepSpinminiprepkit.ThiskitusesacolumnwhichbindstheDNAfromcelllysate inahigh-saltbuer,thenelutesitinlow-saltbuerProtocolsummarizedinFigure27.The amountofDNApresentinthesampleswasdeterminedbymeasuringtheabsorptionat260nm 16

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usingaspectrophotometer. Adoubledigestwasthenperformedoneachplasmid,inordertoconrmthepresenceofthe desired mdh-1 insertintheplasmid.Thedigestswereconductedusingtherestrictionenzymes XbaIandXhoI.Theserestrictionenzymeswerechosentocutoutthe1Kb mdh-1 genefromthe remainingplasmidvector,leavingalinearvectorofabout6Kb.Thelocationoftheserestriction sitesinthepTXB1plasmidcanbeseeninFigure10.ThereactionrecipecanbeseeninTable 2.EachdigestwasconductedIna0.5mLeppendorftubefor1hourat37 Table2:DoubleDigestReactions. PlasmidDNAfrom10cellcultures,S1-S5andL1-L5were cutwithrestrictionenzymesXho1andXba1. PlasmidDNA 2 L NEBBuer#4 1 L BSA10Xbuer 1 L Xho1 0.5 L Xba1 0.5 L NPH 2 0 5 L AgarosegelelectrophoresiswasusedtoseparateDNAfragments.OrangeGloadingdye Lwasrstaddedtoeachdoubledigestsample,andthentheentire12 Lsolutionwaspipetted intoitsrespectivewellina1%agarosegel.A1Kbladderwasplacedinthemiddlewell.An electriccurrentwasappliedfor40minutesat150V,andthenegativelychargednucleicacids movedthroughthegeltowardthepositiveelectrode,separatingDNAfractionsbysizesmaller, lighterfragmentsmovefaster/fartherthanlongfragments.Auorescentdye,GelRed,was addedtotheagarosegelforUVdetectionoftheDNAbands.ThegeluorescedunderUVlight buttheDNAbandscreateddarkshadows. Chemocometentcellsprepared Chemocompetentcellswerepreparedasahostforthepreparedplasmids.ER2566cellswere removedfromthe-80 Cstoragefreezerandsmearedwithaame-sterilizedwirelooponto LBplates.Theywereincubatedat37 Covernight.Two5mLLBcultureswerebegunand placedintheshakerat37 Covernight.Thecultureswerethendiluted1:100,0.25mLin25 mLofLB,andplacedin125mLerlenmeyerasks.Theywereincubatedintheshakerfor approximately21/2hoursandcheckedwithaspectrophotometeruntilOD 500 =0.4.Thecells werespundownat4 Cfor10minutesat4000rpm,andtheLBbrothwasdiscarded.The cellpelletwasresuspendedin12.5mLof50mMCaCl 2 solutionandincubatedonicefor30 minutes.Thecellswerecentrifugedasbefore,andthesupernatantdiscarded.Thecellswere thenresuspendedagainin2.5mLof50mMCaCl 2 solutionand50 Lwasaliquotedintofour 1.5mLeppendorftubesfortransforming.Glycerolwasaddedtotheremainingcellstomakea 17

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10%glycerolsolutionabout0.25mLandtheywerealiquotedinto20,1.5mLeppendorftubes, 50 Lpertube.Thesecellsampleswerequickfrozenwithliquidnitrogenandstoredinthe-80 Cfreezer. TransformationandPreparationofStockCells TransformationswereperformedusingthepreparedpTXB1plasmidswith mdh-1 inserts, andcellswereculturedtoprepareforasmall-scaleinduction.FourtubesofER2566cellswere thawedandlabeledS4,S5,L4andL5.Toeachofthese,1 Lofthecorrespondingplasmid DNAwasadded.Thecellswereheatshockedat37 Cfor1minutetofacilitatetransformation, thenrecoveredin0.5mLSOCmediaintheshakerat37 Cforonehour.Eachofthefour samplesweregrownonLB-ampplatesovernight Land100 Lplateswerespread.All platesgrewsuccessfully.Foreachclone,onecolonyofcellswastransferredfromtheplateinto 5mLofLB-ampwithaame-sterilizedwireloop.Thecultureswereincubatedovernightat37 Cwithshaking. Partofthesecultureswerestoredasstockcells.Ina1.5mLeppendorftube,0.85mLof eachculturewasaddedto0.25mLglycerolandquick-frozeninliquidnitrogen.Thesecellswere labeledER2566+pTXB1S4,S5,L4,andL5respectively,andstoredat-80 C. InducingTargetGeneExpression Small-scaleInduction1:SDS-PAGE Afterstockcellswerealiquoted,small-scaleinductionswereperformedonthecultures.Each culturewasdiluted1:100,50 Lofculturein5mLLB-ampbroth,andincubatedwithshakingat 37 C.Thecultureswereexaminedwithaspectrophotometeratincreasinglyfrequentintervals untiltheyhadgrowntoOD 600 =0.5about4hours.TwomLofcellswerespundownandstored at-20 Casanuninducedcontrol.Tobegininduction,2.4 Lof40 g/mLmMIPTG stocksolutionwasaddedpermLofculturetoobtainatotalconcentrationof0.4mMIPTGin eachculturetubetherecommendedconcentrationintheIMPACTkitliterature. Intheabsenceoflactose,the lac operonisinactivatedbythelacrepressor,whichbindsto thelacoperatorandinterfereswithRNApolymerasebinding.Inanormalcell,whenlactoseis introduceditisconvertedtoallolactose,whichbindstotherepressor,deactivatingitandinducing thetranscriptionofthe lac operon.Theoperonincludes lacZ ,thegeneencoding -galactosidase, whichthenbreaksdowntheallolactose.IPTG,orIsopropyl -D-1-thiogalactopyranoside,is anallolactoseanalog,whichbindstotherepressorbutisnotasubstratefor -galactosidase. ThusIPTGconcentrationinthesolutionstaysconstantasitinducestranscriptionofthelac 18

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operon.IntheER2566 E.coli strain,the lacZ geneisreplacedwiththeT7RNApolymerase geneapolymerasewhichishighlyselectivefortheT7promotersequence;thuswhenIPTGis introducedtothegrowthmedia,itinducesexpressionofT7RNApolymerase.WhenthepTXB1mdh-1 vectorispresent,theT7RNAPbindsselectivelytotheT7promoterpositionedupstream fromthe mdh-1 gene,resultinginhighlyspecictranscription,andsubsequentexpressionofthe targetprotein.ThelocationoftheT7promotercanbeseeninFigure10. AftertheintroductionofIPTG,theinductionswereincubatedintheshakerat37 C.After 2hours,40 Lwereremovedandplacedintoaneppendorftubewith10 Lof6XSDSbuer. Thesewerelabeledashrinduced"andstoredat-20 C.At3hoursafterIPTGwasadded, another40 Lwereremovedandplacedintoaneppendorftubewith10 Lof6XSDSbuer. Thesewerelabeledashrinduced"andstoredat-20 C. ThesampleswereanalyzedviaSodiumDodecylSulfate-PolyacrylamideGelElectrophoresis, orSDS-PAGE.SDSisadetergentwhichlinearizesproteinsandcoversthemwithnegative charges,leavingallproteinswithanequalcharge:massratio.Theproteinsarethenrunthrough anacrylamidegelbyanelectriceld;shortproteinsmovemorequicklytowardtheanodeatthe bottomofthegel,sincetheyexperiencelessfrictionwithinthepolymermatrix.Thegelincludes aresolvingportionwhichseparatestheproteinsintodierentbands,andaloadingorstacking portionwhichhelpstheproteins`lineup'beforeenteringtheresolvingportion.Thesolutions fortheappropriatebuersandtheacrylamidegelsareincludedintheappendix.Extracareis takenwhenhandlingacrylamide,asitisaneurotoxin. Eachuninduced,twohour,andthreehoursamplewasboiledfor5minutesbeforeloading 8-10 Lofeachintoitsrespectivewellintheprepared10%acrylamidegel.Thegelwasrunat 200Vand35mApergel;70mAfortwogels,forabout45minutesoruntilthebromophenol bluedyeintheSDSbuerreachesthebottomofthegel.Thegelswereremovedfromthe apparatus,rinsedwithDIwaterandstainedwithGelCodeBlue.Itwasdecidedthatawestern blotwasnecessaryinordertodetermineiftheinductionwassuccessful. Small-scaleInduction2:WesternBlotting OvernightculturesoftheL4,L5,S4andS5clonesweregrownandthesmall-scaleinductions wererepeated.Theuninduced,twohour,andthreehoursampleswereboiledandloadedintoa standardSDS-PAGEsetup.Whenthegelwasdone,awesternblotwasperformed.Thistechniquetransferstheproteinbandsontoamembraneandprobesthemusingimmunodetection. Thegelwasremovedfromtheglassplatesandlaidontopofapieceof3MMpaper.Meanwhile,theImmobilon-PwesterntransfermembraneMillipore,madeofPolyvinylideneFluoride PVDF,wascuttosize.Themembranewaswashedinmethanolforafewseconds,thenwashed 19

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withwaterandequilibratedwithwesterntransferbuer.Themembranewasthenplacedon topofthegelandsmoothedoutwithaglassrodsothattherewerenobubblesbetweenthem. Anotherpieceof3MMpaperwasplacedontopofthemembraneandwasagainsmoothedout withaglassrod.Spongeswereplacedoneithersideofthesandwichandtheentiresandwich waspressedinacassette.Thecassettewasplacedintoatankwithtransferbuer,makingsure themembranewaspointingtowardapositiveelectrode.CurrentmAwasappliedovernight withstirring.Duringthistime,theproteinbands,stillnegativelychargedfromtheSDS,were forcedtomigratefromtheacrylamidegelontothePVDFmembrane.ThePVDFmembrane wasthenremovedfromthesandwichandsoakedinfoursolutions. TherstsolutionwasablockingsolutionmadefromTTBSTris-BueredSaline/Tween-20 and3%nonfatdrymilk.Itwasappliedtothemembraneforonehourwithshaking.Themilk proteinsboundtotheareasofthemembranethatdidnotalreadycontainproteinbands.The membranewasthenwashedinTTBStwicefor10minutes.Thenextsolutionwastheprimary antibodysolution,a1:5000dilutionofrabbitanti-chitinbindingdomainNEBinTTBSwith 0.5%nonfatmilk,foratleastonehourwithshaking.Theantibodiesinthissolutionbindto thechitinbindingdomainoftheinteintagontheMDH-1aswellasanyotherchitinbinding domainspresentinthelysedcellcontents.Theprimaryantibodysolutionwassavedandstored at4 C.Itcanbeusedfor3-4westernblots.ThemembranewasagainwashedwithTTBStwice fortenminutes.Thenextsolutionwasthesecondaryantibodysolution,a1:7000dilutionof anti-rabbitIgGPromegainTTBSand0.5%nonfatmilk,foratleastanhourwithshaking. Theseantibodiesbindtheanti-CBDantibodies.Themembranewaswashedthreetimesin TTBSfortenminutes.Thefourthandnalsolution,APbuer,wasthedevelopingbuer.The secondaryantibodycontainsanAlkalinePhosphatasedomainwhichreactstotheBCIPand NBTintheAPdevelopertocreateapurple/brownstainatthesiteoftheproteinbandsin question.Inthiswesternblot,therewereclearinducedbandsbuttheladderwastoofaintto detectthesizeofthebands. Allappropriatebuerandwashsolutionsareincludedintheappendix. Small-scaleInduction3:OptimizationofExpression Athirdsmall-scaleinductionwasperformedwithvariedexpressionconditions.FreshL4and S4stockcellswerestreakedonLB-ampplatesandincubatedat37 Covernight.Colonieswere transferredtoculturetubeswith5mLLB-ampbroth,andculturesweregrownovernightat37 C.Induction3proceededasthepreviousinductions,withthefollowingalterations.Thecells werediluted1:100into5mLofTerric-ampbroth.Terricbrothisamoreconcentratedmedia preparationthanLB.HalfofthecellswereinducedafterincubatingtoanOD 600 of0.5,and 20

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theotherhalfwereinducedatanOD 600 of1.UninducedsamplesweretakenatbothOD 600 valuesbeforeIPTGwasadded.Fortheinducedsamples,1mLwasremovedfromtheculture andaddedto1mLofpre-warmedterric-amp-IPTGbrothandincubatedat37 C.Samplesof 1mLwereremovedfromeachinducedcultureattwoandfourhourtimepoints,spundownand storedat-20 C.Thesampleswerethawedandresuspendedin20 Lof5XSDSbuer.The sampleswerethenboiledfor5minutes,and10 Lofeachwasloadedontoacrylamidegels.The SDS-PAGEandwesternprotocolswerefollowedasbefore.Thesewereusedtodeterminewhich conditionsinducedthestrongestexpression. Large-scaleInduction1andPuricationonChitinColumn Theinductionprocedurewasnowconductedonamuchlargerscale.Fromfresh5mL culturesofS4andL4clones,2mLwasdilutedinto200mLofterric-ampbroth.Thecultures weregrowntoanOD 600 of0.5.Afteruninducedsamplesweretaken,IPTGwasaddedtoa concentrationof0.4mMandcellswereinducedforthreehours.Thecellcultureswerethen spundownfor10minat4000gat4 C,andstoredat-20 Covernight.IMPACTprotocol wasfollowedforthesingle-columnpurication.Cellpelletswereresuspendedin20mLcolumn buerwith0.8mLproteaseinhibitorsRoche,25X,andlysedbysonicationfor5minutes secondson/15secondsrestcycles.Thelysedcellswerecentrifugedat15000rpm,4 C,for 30minutestopelletunwantedcellcomponents.Thesupernatant,nowtheclariedcellextract, wasreadytobeappliedtothechitincolumn.Twocolumnswerettedwith2mLofchitinbead slurry,whichisabout50%chitinbeads.ThecolumnswerelabeledL4andS4.Eachcolumnwas equilibratedwithcolumnbuerbyrunning8mLthroughthecolumn.Theclariedcellextracts wereappliedtotheirrespectivecolumnsatabout1mL/min,tobindtheMDH-intein-CBD. Thecolumnswerewashedwith30mLofcolumnbuer.Cleavagebuerwaspreparedfreshlyby adding0.5mLof1MDTTto10mLofcolumnbuer,foraworkingDTTconcentrationof50 mM.About5mLofcleavagebuerwasquicklyrunthroughthecolumn.Thecolumnowwas thenstopped.Bothcolumnswereleftovernightat4 Ctoallowinteincleavagetooccur.The columnsweretheelutedusingcolumnbuer.Three1.5mLelutionfractionswerecollectedfrom eachcolumn.Thecolumnswerethenstripped.First,20mLstrippingsolutionwasrunthrough eachcolumn,followedby40mLwaterand10mLcolumnbuertoregeneratethecolumns. Noproteinwaspresentintheelutionsamples.Uponfurtherexamination,itseemedthere werenosignicantdierencesbetweenbandspresentintheinducedsamplesandtheuninduced samples,indicatinganoverallweakinduction. 21

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Largescaleinduction2 Thelarge-scaleinductionmethodwasrepeated.Fresh200mLculturesweregrownto OD 500 =0.5andinducedwith0.4mMIPTG.Afterthreehoursofincubationat0.4mMIPTG, thecellswerespundownandstoredat-20 C.Uninducedandinducedculturesamplesof0.5mL werespundownattwoandthreehourtimepointsforanalysis.Itwasdesirabletorstverifythe successoftheinductionbeforerepeatingthecolumnpuricationprocedure,sotheinducedand uninducedsampleswererunonSDS-PAGE.Eachcellpelletwasmixedwith50 Lof1XSDS loadingbuer.Thesampleswereboiled4minutesbeforeloadingontotheacrylamidegel.The gelswerestainedwithcoomassieblue.Theyshowedveryweakbandsintheinducedsamples comparedtotheuninduced. Site-directedMutagenesisofInteintag/MDH-1interface UponexaminationoftheIMPACTliterature,itseemedthatthedesignofthe mdh-1 -intein interfacewasnotconducivetoecientinteincleavageonthechitincolumn,duetotheamino acidwhichwasdirectlyadjacenttotheintein.TheIMPACTsystemprovidesinformationasto whichaminoacidscleavemoreeectively.Untilthispoint,aserinewasadjacenttotheintein, whichcorrespondsto5-15%cleavageeciency.Itwasdecidedthattheseserineresidueswould bereplaced,viasitedirectedmutagenesis,toensurebettercleavageonthechitincolumn. Thesite-directedmutagenesiswasconductedusingtheQuikChangeSite-DirectedMutagenesisKitStratagene.TheoverallschematicforthisprocedureisshowninFigure11.Themutagenesisincludedsinglestrandedprimers,doublestrandedtemplateDNA,adNTPmixture, andaDNApolymeraseinbuer.Theexistingdouble-strandedplasmaDNAwasdenatured withheat,thencooledtoallowmutagenicprimersshortDNAfragmentscontainingdesired mutationstoannealtoeachstrand.Oncetheprimersannealed,thepolymeraseelongatedthe strands,completingeachstrandoftheplasmid.Thesolutionwasheatedagain,thestrands againseparatedonestrandcontainingthemutation,thentheprimersannealedagain,and thecyclewasrepeated.Afterseveralcycles,themutatedcopiesoftheplasmidsfarexceedthe nativecopies. PrimerswerechosenbasedonguidelinesfromboththeIMPACTkitandtheQuikChange Site-DirectedMutagenesisKit.Theprimerswere25-45bplongwiththedesiredmutationin thecenteroftheoligo,10-15correctbasepairsoneitherside,andG/Contheends.Primers haveaminimumof40%GCcontent,andmeltingtemperaturesT m lowerthan78 C. Figures12and13illustratethedesiredmutations.Fortheshort mdh-1 clone,the mdh-1 geneendedatVAF.Thenextsequence,LEGSS,isfromthepolylinkerofthepTXB1plasmid, 22

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Figure11:SiteDirectedMutagenesisSchematic Step1:DNAwasusedfromPlasmidPreparationsofL4andS4clones.Step2:Single-strandedprimersweredesignedwhichcontained desiredmutationsSeeFigures12and13.Temperaturecyclingwasperformedtodenature DNA,annealprimers,andelongatestrands.Step3:NativeplasmidDNAnon-mutagenicwas digestedbytheenzymeDpnI.Step4:MutatedplasmidDNAwastransformedinto E.coli cells. Copyright2003,Stratagene. 23

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thenCITGisthestartpointfortheinteinsequence.Aprimerwasdesignedtomutatethe polylinkeroftheplasmidtoremovetheLEGSSsequenceandreplaceitwiththenatural mdh-1 sequence,VKGN.Asnisanadequateaminoacidforthepositionadjacenttotheintein,asit produces65-80%cleavageafter16hoursat4 C.Inthe mdh-1 longclone,the mdh-1 gene endedattheVAFVKGNsequence,wheretheLEGSSpolylinkersequencebegan.ThelastSerin LEGSSwasthe'problem'aminoacid,whichwasmutatedintoanAla.AccordingtoIMPACT kitliterature,Alaproduces65-80%cleavage. Figure12:SiteDirectedMutagenesis,ShortGene TheLEGSSsequencewasremovedandreplacedwiththenaturalmdh-1sequence,VKGN,leavinganAsnadjacenttotheinteinseuqence. Figure13:SiteDirectedMutagenesis,LongGene WithCITGasthestartpointfortheintein sequence,thelastSerinLEGSSwasthe'problem'aminoacidthatwaschangedtoAla. GelPuricationofMutagenicPrimers Theprimersforboththeshortandlongclones,alongwiththeirreciprocalsequences,were orderedfromanoutsidelabandgel-puriedbeforeusingthemforthemutagenesisreaction. First,5mMTEbuerwasaddedtothedryoligostobringthemto300 M.Then75 Lof 300 Mprimerwasaddedto75 Lof2XUrealoadingbuer.Thesampleswereheatedto80 Cforabout5minutes,thenloadedintolargewellsina10%acrylamide-ureagelandrunat 70mAfortwogels,200V,forabout35minutes.TheamountofAPSusedtopolymerizethe 24

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acrylamidewasdoubledfrom35to70 L,astherewassometroublewiththegelssolidifying. TheTEMEDwasalsoincreased,from15to25 L,todecreasethepolymerizationtime.When electrophoresiswascomplete,eachgelwasremovedfromtheelectrophoresisapparatus,placed onsaranwrap,andlaidontopofaTLCplatewithauorescentindicator.UnderUVlight, theDNAlookslikeadarkbandinthegel,becausetheDNAblocksthelight.EachdarkDNA bandwascutoutofthegelwithascalpelandplacedina1.5mLeppendorftube.Carewas takenwhencuttingthegelsothatsmallerpiecesdidnotgetinwiththemainband.Thegel wascrushedupwithapipettetipand0.4mLofgelelutionbuerormorewhennecessary wasaddedtothetube.Whenthiswasdoneforallfourprimers,thetubeswereashfrozen inliquidnitrogenandthawed.Thefreeze/thawstepwasrepeated.Thetubesweresealedand tapedtothebottomoftheincubatortoeluteovernightat37 Cwithshaking.Thenextday 0.3mLofphenol-chloroformwasaddedtoeachtube,vortexed,andcentrifugedathighspeed foroneminute.Theaqueouslayerswereputinfreshtubes.Another150 Lofgelelution buerwasaddedtothephenollayers,vortexedandspuntoelutemorefromthephenol.These aqueouslayerswereaddedtothepreviousones.Thecombinedaqueouslayerswerespunfor 2minutesathighspeedtoseparatetheremainingphenol/gelpieces.Toeachcleanaqueous layer,1 Lofglycogenwasadded.TheDNAinsolutionwasthenprecipitatedinethanolwith freezing.Ethanolequaltotwotimesthevolumeofeachtubewasadded,andtheoligoswere frozenforfourhoursat-20 C.Theywerethenspunathighspeedat4 Cfor30minutes.The supernatantwasdiscardedandthesmallDNApelletsinthebottomofeachtubewerewashed withethanolanddried.Thepelletswereresuspendedin30 Lof5mMTris,pH8.0.Thefour primerswereanalyzedinaspectondtheirconcentrations,whichrangedfrom180-280 M. Mutagenesis:ThermalCyclingandTransformation Site-directedmutagenesisreactionswereperformedusingthegel-puriedprimers.Afew Lofeacholigowasdilutedwith5mMTristomakea3 Msolution.Abitofeachofthe S4andL4plasmidDNAstocksolutionsweredilutedto5ng/ LinnpH 2 O.Themutagenesis reactionsolutionsaredetailedinTable3.Afterthesolutionswereprepared,1 LofPfuUltra DNApolymerasewasaddedtoeachandtheywereruninthePCRmachineonprogramMUT18 Step2,Figure11.Thisprogramheatsthesolutionsto95 Cfor30secondstodenatureDNA, thencoolsto55 Cfor1minutetoallowtheDNAprimerstoanneal,thenbringsthesolutions to68 Cfor7minutestoallowelongationofthestrands.Thiscyclethenrepeats18timesto ensuresucientamplicationofthemutatedDNA.Themutagenesisreactionswerethenplaced inthe-20 Cfreezerovernight. Themutagenesisreactionswerethendigestedwith1 LofDpnIat37 Cfor1hour.Step 25

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Table3:SiteDirectedMutagenesisReactions.DNApolymerasewasaddedabsolutelylast. S4,10ng S4,20ng L4,10ng L4,20ng S4plasmid ng=L 2 L 4 L L4plasmid ng=L 2 L 4 L Sprimer M 5 L 5 L SRCprimer M 5 L 5 L Lprimer M 5 L 5 L LRCprimer M 5 L 5 L 10XPCRreactionBuer 5 L 5 L 5 L 5 L dNTPmixture 10 L 10 L 10 L 10 L npH 2 O 23 L 21 L 23 L 21 L PfuUltraDNAPolymerase 1 L 1 L 1 L 1 L 3,Figure11.TheDpnIenzymedigestsonlythenativeplasmidDNA,whichwasmethylated. Thisdigestthusremovesthenon-mutatedcopiesoftheplasmidsothatonlythosewiththe desiredsequenceremain. ThedigestedPCRproductwastransformedinto E.coli cellsStep4,Figure11.Electrocompetentcellswereremovedfromstorageat-80 Candsplitintofourtubes,eachwith25 Lofcells,labeledS410,S420,L410,L420.TheappropriateDpnIdigestreactionwasadded toeachtube Laliquotsandthecellsweretransferredinto1mmcuvettes.Thecellswere electroporatedat1.7kV,25 F,and200.Thecellsrecoveredin0.5mLSOCmediaat37 C foranhour.EachtransformationsamplewasspreadontothreeLB-ampplates,inaliquotsof 50,150and300 L,withaame-sterilizedglassspreader.The50and150 Lplateswerespread rst.Thentheremaining300 Lwasspundown,and200 Lofsupernatantwasdiscarded. Thecellswereresuspendedintheremainingsupernatant,andspreadontheplate.Theplates wereallowedtosetforafewminutes,theninvertedandplacedintheincubatorovernightat 37 C.Inthemorningtheplateswereremovedfromtheincubatorandcolonieswereselected forculturing.FiveS410,S420,andL410colonieswerecultured,aswellasthreeL420colonies therewereonlythreesuccessfulcoloniesontheagarplates.ThecultureswerelabeledS410 A,S410B,etc.Theywereculturedin5mLLB-ampbrothovernightat37 Cwithshaking. AllL410andL420coloniesgrew,aswellasoneS410colonyS410A.TheremainingS410and allS420coloniesfailedtogrow.Allsuccessfulcultureswerespundownandstoredat-20 C. ScreeningforMutatedClones TheQIAGENminiprepplasmidprepprotocolwasfollowedtoextracttheplasmidDNA foreachoftheL410A-E,L420A-C,andS410AcellsProtocolsummarizedinFigure27. TheprepswerequantitatedintheCary-14spectrophotometer,andconcentrationsrangedfrom 150-450ng/ L. Theplasmidprepswerethentestedwithadoubledigestreactiontoseeifthedesiredmu26

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Table4:DoubleDigestReactions. PlasmidDNAfrommutatedcloneswerecutwithrestriction enzymestoconrmsuccessfulmutations. Ligation: Short Long NEBBuer#4 1 L 1 L BSA10Xbuer 1 L 1 L XhoI 0.5 L XbaI 0.5 L 0.5 L SapI 0.5 L S410ADNA 3 L [L410A-E,L420A-C]DNA 3 L NPH 2 0 4 L 4 L tationswerepresent.ThedoubledigestrecipescanbeseeninTable4.Eachplasmidwascut withonerestrictionenzymetolinearizetheDNA,andthenanotherrestrictionenzymewhich wouldcuttheoriginalsequenceofDNAatthelocationofmutation.Ifthissequencehadbeen mutated,thesecondenzymewouldnotcuttheDNA,andthelinearizedplasmidwouldremain intact.Fortheshortplasmid,thisincludedrestrictionenzymesXbaIandXhoI.Forthelong plasmid,XbaIandSapI.ThetargetedrestrictionsitescanbeseeninFigure10. Alldigestswereassembled,placedinthePCRmachine,andrunat37 Cfor1hour.They werestoredat-20 Covernight.Orangegdye Lwasaddedtoeachofthedigestsand theywerethenrunon1%agarosegelsfor50minat150V.Anotherdigestwascompletedasa positivecontrol;thisdigestincludedtheS410A,L410EandL410Cclones,thistimealongthe originalL4andS4plasmids. UponviewingthegelsunderUVlight,twobandsappearedintheS410Alanes.Thisindicates thattheshortmutationwasunsuccessful.FurtherexperimentationonScloneswasnotpursued. InductionofMutatedClones TheL410EandCcloneswerethentransformedintochemocompetentcells.TwoL410E labeledLE1and2andtwoL410ClabeledtoLC1and25mLLB-ampculturesweregrown overnight,diluted100X,60 Lin6mLLB-ampbroth,andincubateduntilOD 600 =0.5. Uninducedsampleswereincubatedseparately.IPTGwasaddedtoanalconcentrationof 0.4mMinLE1andLC1,and0.8mMinLE2andLC2.Afterthreeandfourhours,0.45mL samplesofeachculturewerespundown.EachcellpelletwaspreparedforSDS-PAGEbyadding 45 L1XSDSbuerandboilingfor4minutes.Gelswerepreparedandallsampleswereloaded, witheachuninducedsamplenexttoitsrespective3and4hoursamples.Thegelswerestained withcoommassieblue. AlargemLLB-ampinductionwasthenconductedasbefore,at0.4mMforthreehours. Allthecellswerespundownat4 C,withsmallinducedanduninducedsamplesreservedfor analysis.Thechitincolumnprocedurewasthenrepeatedasbefore.Thefollowingsampleswere 27

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takenforanalysisbygelelectrophoresis:inducedclariedsupernatantaftercellswerelysed, centrifuged,cellpelletaftercentrifugation,columnowthrough,fourelutionfractions,early wash,latewash,quickDTTush,columnresinbeforeDTT,columnresinafterelution. Nodetectableproteinwaselutedfromthecolumn.Thecolumnwasstrippedaccordingto protocol. ComparingMutated,Non-mutatedandNo-insertplasmids Comparinginducedtouninducedsamples,alargeinducedbandwasapparentatabout30 kDa.TheMDH-1proteinattachedtotheinteintagshouldamounttoamuchlargerprotein, about60-65kDa-35fortheMDH-1,about28kDafortheinteintag.However,aninduced bandatthissizewasnotapparent.Severalpossibilitieswereexploredinordertondthesource ofthe30kDaband. First,8small-scaleinductionswereperformedtoseeifthesizevariedbetweenthemutated clones,andtodetermineifanyofthemcontainedunforeseenmutations.L10A-EandL20A-C plasmidswereeachtransformedintochemocompetentER2566 E.coli cells.LB-ampculturesof eachcloneweregrownovernight.Astandardsmall-scale3-hourIPTGinductionwasperformed onall8clones.Duplicate1mLuninducedandinducedsampleswerespundownandanalyzed viaSDS-PAGE. Allclonesproducedthesame30kDaband.Itseemedpossiblethattheinteintagwas cleavingtoosoon,andthebandrepresentedeitherthetagitselforthenativeMDH-1they wereapproximatelythesamesize.ThiscouldhavebeencausedbytheDTTinthesample buer.SparepelletsfromapreviousL4inductionandanL410Cinductionwereanalyzedvia SDS-PAGEandwesternblot,withandwithoutDTTinthebuer,buttherewerenosignicant dierencesbetweenthesamples. AnotherpossibilitywasastopcodonwithintheMDH-1sequence,cuttingtheexpressed proteinshort.SamplesofplasmidDNAwerepreparedtobesentforsequencing.Sinceall mutatedL4'slookedidentical,onlyL410Cwassenttobesequenced.OriginalL4andS4samples weresentaswell.Samplespreparedwereeach30 Linvolume,containing100ng/ L.The sequencesdidnotindicateanymutations. Finally,the30kDabandmaynothavebeenMDH-1,butanotherproteinaliatedwith theinduction.TheoriginalpTXB1plasmidnoinsertandL410Cplasmidweretransformed intochemocompetentcellsandonecolonyofeachwasculturedinLBovernight.Astandard small-scale3hour,37 C,0.8mMIPTGinductionwasperformedonboth,inadditiontoa longerroomtemperatureinduction.Forthelonginduction,5mLoffreshlygrowncellsODof 28

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0.5wereinducedwith0.8mMIPTGandshakenat200rpmfor12hoursatroomtemperature. Forbothinductions,uninducedsampleswereincubatedseparately.Three1mLsamplesof eachuninducedandinducedfractionlongandshortwerespundownforanalysis.Thecell pelletswereresuspendedin100 L1XSDSbuerandboiledfor3-5minutes.Thecellsamples becameveryviscousafterboiling,duetotheDNApresentinthesample.Thiswasalleviatedby runningthesamplesthrougha25gaugeneedlewhilestillwarm,inordertobreakuptheDNA fragments.Thesampleswerethenrunontwoidenticalgels;onewasstainedwithcoomassie blueandonewastransferredanddevelopedinawesternblot.The30kDabandwaspresent inboththeL410CandtheNo-insertsamples.ThisindicatesnoaliationbetweentheMDH-1 andthe30kDaband.Ifthe mdh-1 genewasbeingexpressedinthesecells,itmaynothave beeninquantitieslargeenoughtodetectviaSDS-PAGEorawesternblot. FinalPuricationonChitinColumn Despitethelackofa65kDabandpresentintheinducedsamples,anotherchitincolumn puricationwasconducted,withaslightlymodiedprocedure.Three-hourinductionswere performedin750mL2:1LB:TB,usingcellscontainingoriginalL4plasmidandtheL410C mutatedplasmid.Thecellswerespundownat4 Candstoredat-20 C.Pelletscontaining 200mLofL4cellsand200mLL410CabbreviatedLCcellswereeachresuspendedin5mL celllysisbuer.Theywerelysedbysonicationfor3minutesin15secondson/15secondsrest cycles.Aliquotsof40 Lofeachcrudecelllysissolutionwereaddedto20 L3XSDSsample buerandstoredasa`crude'sample.Allsamplesduringthefollowingprocedureweretakenin duplicate.Thecellswerethencentrifugedfor30minutesat4 Cand10000rpm.Unwantedcell componentswerecollectedinapelletatthebottomofthetube,andtheclariedcellextract containingtheMDH-1remainedasthesupernatant.Samplesofeachclariedcellextract Lin20 L3XSDSsamplebuerwerestoredasa`clar'sample.Theremainderoftheclaried cellextractwasthenreadytobeappliedtothechitincolumn.Thepelletswereresuspendedin 1mLofcolumnbuer,and40 Lwasaddedto20 L1XSDSbuerasa`pel'sample.This samplewouldrevealanysolubilityproblems. Twocolumnswereeachttedwith6mLofchitinbeadslurryandlabeledLC"andL4". Abouttenvolumes-60mLofcolumnbuerwasrunthrougheachcolumnbeforeadding thecelllysate.Theclariedcellextractsabout5mLeachwereloadedontotheirrespective columnsatarateofabout0.5mL/min.Samplesofcolumnow-throughfromeachcolumnwere collected Lin20 L3XSDSsamplebuerandstoredas'FT'samples.Onceallofthe clariedcelllysatewasloaded,eachcolumnwaswashedwith50mLofcolumnbuer,atabout2 mL/min.Washsamplesweretakenfromeach.Firstresinsampleswerealsocollectedbyadding 29

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100 Lresinto50 L3XSDSbuer.Theelutionbuerwaspreparedfreshlyduringthewashes, byaddingDTTtocolumnbuer.AboutthreevolumesmLofelutionbuerwasquickly appliedtoeachcolumn,about5mL/min.Someofthisow-throughwascollectedforsamples labeledQF"quickush.Thecolumnowwasstoppedandtheinteincleavageproceededfor 18hoursat4 C.Thecolumnswerethenelutedwithabout5mLofcolumnbuer,1mL/min. Five1mLelutionfractionswerecollectedandlabeledE1-E5foreachcolumn.Fortheelution samples,40 Lofeachelutionweremixedwith20 L3XSDSbuer.About20%Glycerolwas addedtotheelutionfractionsandtheywerestoredat-20 C.Secondresinsamplesweretaken asbefore.Columnowwasstoppedandthecolumnswerestoredat4 C. Twogelswererunforeachclone,L4andLC.Onegelcontaineduninduced,crude,pel,clar, FT,Res1,wash,QF,andRes2samples.ThesecondgelcontainedUninduced,clar,FT,and E1-E5samples.Thesegelsweretransferredtowesternmembranesandtheblotsweredeveloped thefollowingday.Aduplicatesetofgelswererunandstainedwithcoomassieblue. Enzymeassays Fromthestainedacrylamidegels,theelutionfractionswiththemostconcentratedMDH-1 couldbedetermined.Usingthesefractions,assayswereconductedtodeterminetheactivityof theMDH-1inthesamples.IntheKrebscycle,MDH-1usesNAD + asacoenzyme,convertingit intoNADHwhileconvertingmalateintooxaloacetate.Thisreactionisenergeticallyunfavorable, butitisabletoproceedinthecontextofthecellbecausetheoxaloacetateisusedquicklyafterits formation.Forenzymeassays,itwasmoreconvenienttousethereversereaction,theconversion ofoxaloacetateintomalate.BecauseNADHabsorbslightat340nm,whileNAD + doesnot, theprogressionofthereactioncouldbeeasilyanalyzedbymonitoringthedropinabsorbance at340nm. Preliminaryassayswereconductedrsttondoutiftheproteinpresentinthesample wasindeedactive.First,150mMoxaloacetateand60mMNADHstocksolutionsweremade. Theseconcentrationswereatleast10Xtheconcentrationneededfortheassays.Thesereagents degraderatherquicklyandthusneededtobemadefreshforeachbatchofassays.Theywere keptonice,asweretheproteinsamples,andtheNADHwascoveredintinfoilsinceitis photosensitive.Theassaybuerwaskeptatroomtemperature,andthustheassaysthemselves wereconductedatroomtemperaturesincethemajorityoftheassaycontentwasthebuer. Therst0.5mLreactionscontained50 LNADH M,50 Loxaloacetate M,and 380 Lofkineticassaybuer,whichwas0.1Mpotassiumphosphate,pH7.5,and0.1MKCl. Tothismixture,20 LoftheE3samplefromthechitincolumnwasadded.Thecomplete 30

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reactionmixturewasimmediatelypipettedintoaquartzcuvetteandplacedintotheCary-14 spectrophotometer.Thesolutionwasscannedforabsorbanceat340nmfor4minutes.This reactionshowedveryhighactivity,sotheremainingassayscontainedonly10 LofMDH-1 sampleand10 Lofadditionalkineticassaybuer. AssayswereperformedwithseveralotherconcentrationsofOAA,between0-150 M.Each absorbancevs.timegraphwasanalyzedinOriginOriginLab.Theywererstttoexponential decays,thenthederivativeofeachequationwastakentondtheinitialrateofreactionfora given[OAA].V 0 vsOxaloacetateconcentrationwasplottedonagraphtodetermineK m and V max DialysisandProteinConcentrationAssay TheenzymesamplesweredialyzedtoremovetheDTTwhichelutedwiththeproteinfromthe chitincolumn.Dialysisneededtobeperformedbeforetheconcentrationoftheproteinsamples couldbedetermined,astheDTTwouldhaveinterferedwiththetests.Onlythefractions LCE1-E4andL4E2,E3weredialyzed.Foreachelutionsample,apieceabout4inofcellulose dialysistubingwascut,foldedandclampedatoneend.Thedialysistubinghasamolecular weightcutoof10kDa,meaningthatallmoleculeslargerthanthisareretained.Eachelution samplewascarefullypipettedintothetubing,andtheotherendwasfoldedandclamped.The secondendwasclampedsuchthatabubblewasstuckineachsample,toensurethattheywould oat.Allsampleswereplacedina1Lbeakerwith500mLofcolddialysisbuer.Thedialysis wentovernightat4 Cwithstirring.Eachpacketwasthenunclamped,returnedto1.5mL eppendorftubes,andstoredat-20 C. Afterdialysis,assayswereconductedtodeterminetheproteinconcentrationineachdialyzed elutionfraction.TheanalysiswasperformedusingtheDCproteinassayBio-Rad,akitmade forcolorimetricproteinquantitation.Thekitworksviaatwo-stepreaction.First,copperreacts withtheproteininalkalinemediumreagentA.Thecopper-treatedproteinreducestheFolin ReagentinreagentB,resultinginacoloredproduct.Thecoloriscausedbythearomaticsidechainsoftryptophanandtyrosine,whichareoxidizedinthereaction.Eachassaywasdonein a96-wellplate.Proteinstandardswithconcentrationsof0.2,0.5,0.8,1.1,and1.5mg/mlwere preparedindialysisbuer,usingbovineserumalbumin.Foreachstandardandsample,5 Lof proteinsolutionwasplacedinthewellrst.Thiswasfollowedby25 LofReagentA,and200 LofReagentB.Afterwaiting15minutes,theplatewasreadinthemicroplatereaderBioTek andanalyzedusingtheGen5softwareBioTek.Theproteinconcentrationwasdeterminedby measuringthesolutions'absorbanceat750nm. 31

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Results&Discussion Summary The mdh-1 genewasligatedintothepTXB1plasmidvector,adjacenttoaninteintagwith achitinbindingdomain.Thevectorwastransformedinto E.coli cellsandexpressionwas inducedusingIPTGIsopropyl -D-1-thiogalactopyranoside.UsingSDS-PAGEandwestern blots,expressionoftheMDH-1-inteinproductwasdiculttodetect.Despitethis,asinglecolumnpuricationschemewassuccessfullyusedtoisolatetheMDH-1.Theconcentration,K m V max ,andspecicactivityoftheelutedproteinweredetermined. LigationofMDH-1intopTXB1andtransformationinto E:coli cells Initially,thetransformationofthepTXB1-mdhvectors,ShortandLong,intothe E.coli cells didnotwork.TheDH5 E.coli cellsweretestedandwereindeedcompetentcells,indicating thatthesourceofdicultywastheligationreactionofthe mdh-1 intothepTXB1vector.This dicultywasalleviatedbyusingafreshtubeofT4DNAligase.After5Sand5Lcolonieswere cultured,plasmidprepswereperformed.Doubledigestsrevealedthepresenceorabsenceofthe desired mdh-1 insert.ThedigestswereconductedusingtherestrictionenzymesXbaIandXhoI. Theseenzymesshouldcutoutthe1Kb mdh-1 genefromtheremainingplasmidvector,leaving alinearvectorofabout6Kb.ThelocationoftheserestrictionsitesinthepTXB1plasmidcan beseeninFigure10.Iftheinsertwasnotpresent,afragmentofabout70bpwouldbecutfrom thegel,whichwouldlikelybetoosmalltodetect. TheagarosegelsresultingfromthedoubledigestreactioncanbeseeninFigure14.The linearizedpTXB1DNAranasathickbandtowardsthetopofthegel,andwaspresentinall lanesexceptS3.Allremainingclones,exceptS5,containthe1.2kBbandrepresentingthe mdh-1 insert.Theinsertshouldhaverunat1kB,butthisdiscrepancycouldbecausedbybuers,the gelortheladderitself,anditwassmallenoughtobeignored. 32

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Figure14:ScreeningPlasmidsfor mdh-1 Insert. Adouble-digestof10plasmidprepsamples,5 shortSand5longLwasconductedusingXhoIandXbaIandrunona1%agarosegel.The desiredinsertisrepresentedbythebandsatabout1.2Kb. InducingTargetGeneExpression ER2566 E.coli culturescontainingpTXB1withtheS4andL4insertswereselectedfor experimentation.Severalsmall-scaleinductionswith0.4mMIPTGwereperformedinorder todeterminethesuccessoftheoverexpressionofMDH-1aswellastheoptimalexpression conditions.First,dierenttime-pointsweretested;cellswereinducedfortwoandthreehours. Celldensityatthetimeofinductionwasalsotested;thecellswereinducedaftergrowingtoan OD 600 of0.5and1.Theresultingwesternblotsshoweddierentbandsintheinducedsamplesas comparedtotheuninduced,butlittleornodierencebetweenthevariedexpressionconditions. Thesizeoftheseexpressionbandscouldnotbedeterminedbecausetheladderwastoofaintto seeonthewesterns[Datanotshown]. Therstlarge-scaleinductionandchitincolumnpuricationwasunsuccessfulandnodetectableproteinwaselutedfromthecolumn[Datanotshown].Anotherlarge-scaleinduction wasperformed,butthistimesmallinducedanduninducedcellsampleswererunonSDS-PAGE beforerepeatingthecolumnpuricationprocedure.Theyshowedveryweakbandsintheinducedsamplescomparedtotheuninduced,anditwasdecidedthatanotherpuricationonthe chitincolumnwouldnotbeperformed.Itispossiblethatthecellsused-S4andL4stockcells, storedat-80 Caftertransformationandculturing-didnothavegoodexpressionbecausethey 33

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werenotfreshlytransformed.Fortheremainderoftheresearch,onlyfreshlytransformedcells wereusedforinductions. Site-DirectedMutagenesis Cleavageeciencyoeredanotherpossibilityastowhytherstpuricationwasunsuccessful. ThedesignoftheMDH-1-inteininterfacecanaectcolumncleavage,duetotheaminoacid locateddirectlyadjacenttotheintein.Untilthispoint,aserinewasadjacenttotheintein sequenceinboththelongandshortversionsoftheMDH-1insert,whichprovidesforonly515%cleavageeciency.Usingsite-directedmutagenesis,theseserineresidueswerereplacedin ordertoensurebettercleavageonthechitincolumn.Detailsaboutthesequencealterations andguresillustratingthechangesareprovidedinthemethodssectionFigures12and13. Theprimerswereorderedandgel-puriedbeforethemutagenesis.Themutatedplasmidswere transformedintochemocompetentER2566 E.coli cellsandplasmidprepswereperformedon successfulcolonies.Theseplasmidprepsweresubjecttoadouble-digesttodetermineifthe mutagenesiswasasuccess.Eachplasmidwascutwitharestrictionenzymetolinearizethe DNA,andthenanotherrestrictionenzymewhichtargetedtheoriginalsequenceofDNAatthe locationofmutation.Ifthemutationwassuccessful,thesecondrestrictionsitewasabsentand thelinearizedplasmidremainedintact.Ifthemutationwasunsuccessful,however,therestriction sitewascleavedandtwobandswouldappearonthegel.Theshortplasmidprepdigestreaction includedXbaIandXhoI,andthe8longplasmidprepdigestreactionsincludedXbaIandSapI .ThetargetedrestrictionsitescanbeseeninFigure10.Theplasmiddigestswererunon1% agarosegelsandcanbeseeninFigure15. AlllaneshadonebandexcepttheS410Alane,whereasecondbandappearedatabout 1.3Kb.ThisindicatedthatthemutationoftheS4clonewasunsuccessful.Thiswasnot surprising,astherewereveryfewScoloniesontheagarplateandonlyoneoftenScultures grewsuccessfully,asopposedtothemutatedLcolonieswhichwereculturedwithoutdiculty. AnotherdigestwascompletedwiththeS410A,L410EandL410Cclones,thistimealongthe originalL4andS4plasmidsFigure16.Theagarosegelinthisexperimentdislodgeditself fromtheapparatus,cuttingtheelectrophoresisshort.Theladderdidnotexpandenoughto gaugethesizeofthebands,butthegelshowedtwobandsintheS4,L4,andS410Alanesand onebandintheremaininglanes.Thisconrmedthepresenceofthesecondrestrictionsitein thenon-mutatedclonesaswellasintheS410Aclone,andthusconrmedtheabsenceofthese sitesinthemutatedL4clones.Thesite-directedmutagenesisoftheLcloneswasasuccess.No furtherexperimentationwaspursuedwithSclones. 34

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Figure15:ScreeningPlasmidsafterSite-directedmutagenesis. Double-digestreactionsofmutatedplasmidprepsamples,1shortS410Aand8longL410A-E,L420A-C,ona1%agarose gel.Twobandsindicatednomutation. Figure16:ScreeningPlasmidsafterSite-directedmutagenesis. Double-digestreactionsofmutatedplasmidprepsamples,1shortS410Aand2longL410A,C,alongsideoriginalS4and L4ascontrols.Runona1%agarosegel.Twobandsindicatesnomutation. 35

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TheL410EandCclonesweretransformedintochemocompetentER2566cellsandcultured. Small-scaleinductionswereperformed.Again,expressionconditionswerevaried;thistime,two IPTGconcentrationsweretested,aswellasthreeandfourhourtimepoints.Gelswererunwith eachuninducedsamplenexttoitsrespective3hrand4hrsamples.TheL410Egelcanbeseen inFigure17. Figure17:Small-scaleinductionofMutatedL410Eclones TestingforIPTGconcentrationand timedependance;LE1cloneswereinducedat0.4mMIPTG,LE2cloneswereinducedat0.8mM IPTG.TotalcellproteinwasrunonaSDS-polyacrylamidegelandstainedwithcoomassieblue. Arrowshowsinducedbandat30kDa. TheinductionwasseeminglysuccessfulinbothL410EandL410Cclones,thoughtherewas nodiscernibledierencebetweentime-pointsorIPTGconcentrations.Themajorinducedband presentwasabout30kDa.Sincethiswasthemostclearlyvisibleinducedband,itwasintuitive toassumethatitwastheMDH-1;however,theMDH-1andinteintagshouldbeamuchlarger protein,about60-65kDa-35fortheMDH-1,about28kDafortheinteintag.Therewas noobviousinducedbandatthecorrectsize. Despitethisissue,alarge-scaleinductionandpuricationwasperformed.Thelargeinduction wasconductedasbefore,at0.4mMIPTGforthreehours.Thecolumnprocedurewasalso repeatedasbefore.Thepuricationwasagainunsuccessful;themainresultsareshownin Figure18.Lanescontainingelutionsamplesshowednoproteinbands.Therewasnosignicant dierencebetweentheclariedextracttotheow-throughsample,indicatingthatnothingbound tothecolumn.Itispossiblethat,ifMDH-1waspresentinthesampleatall,thecolumnmay 36

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haverequiredmorechitinbeadstobindadequateamountsofprotein.Again,the30kDaband appearedintheinducedsamples,withnoclearbandat65kDa. Figure18:ChitinColumnPurication,MutatedLclone. Uninduced=IPTGwasnotintroduced tothesecultures.Induced=Samplecultureswereinducedwith0.4mMIPTGforthreehours. Claried=Supernatantafterinducedcellsampleswerelysedandcentrifugedtopelletunwanted cellcomponents.Flo-thru=columnow-throughduringapplicationoftheclariedlysatetothe chitincolumn.E1-E4=columnelutionsamples,containingnodetectableMDH-1bands.Arrow showsinducedbandat30kDa.Coomassie-stainedpolyacrylamidegel. ComparingMutated,Non-mutatedandNo-insertplasmids Severalpossibilitieswereexploredinordertoexplainthe30kDainducedband,andtotry andndoneat65kDa. Separately,boththeinteintagandtheMDH-1areabout30kDa.The30kDainducedband couldindicatethatsomeoralloftheinteintagswerecleavingtooearly,eitherinvivoorat somepointduringsamplepreparation.SinceSDSbuercontainssmallamountsofDTT,this couldbethecauseofprematurecleavage.SparepelletsfromapreviousL410Einductionwere analyzedviaSDS-PAGEandwesternblot,withandwithoutDTTinthebuer,buttherewas nosignicantdierencebetweenthesamples[Datanotshown].Anotherpossibilitywasthatthe MDH-1wasmutated,causingittobeshorterthanitshouldbepossiblyastopcodoninthe sequence.Inductionswereperformedonallmutatedclonestoseeiftheinducedbandwasthe 37

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samesizeinallcases.ThesampleswereanalyzedwithSDS-PAGEaswellasawesternblot;the westernblotcanbeviewedinFigure19.Theinductionsallappearedtobeidentical.Ineach sampletherewereseveralinducedbands,includingtheunexplained30kDabandandafaint butpromisingMDH-1bandat65kDa. Figure19:WesternblotofSmall-scaleinductionsonallmutatedL4clones. Comparisonof inducedsamplesbywesternblotrevealsthatallcloneswereidentical.LaddermeasuredinkDa. The65kDabandrepresentstheexpressedMDH-1toparrow.The30kDabandremainsbottom arrow. SamplesofplasmidDNAwerepreparedtobesentforsequencing.SinceallmutatedL4's lookedidentical,onlyoneclonewaschosenforsequencing.L410Cwassequencedalongwith originalL4andS4samples.Asequencealignmentforthe mdh-1 genesintheLsamplesisshown Figure20.Nomutationsweredetected,otherthanthedesiredmutationintheL410Cclone. TheS4sequencealsoindicatednomutations[Datanotshown]. Itseemedatthispointthatthe30kDabandwasprobablynottheMDH-1,butmaybe anotherproteinaliatedwiththeinductionprocess.Totestthis,theoriginalpTXB1plasmid noinsertandL410Cplasmidweretransformedintocellsandinducedalongsideoneanother.A standardsmall-scale3hour,37 C,0.8mMIPTGinductionwasperformedonboth,inaddition toa12hour,roomtemperatureinduction.ThesampleswereanalyzedbySDS-PAGEanda westernblot[Resultsnotshown].The12hourinductionproducednoresults.However,the30 kDabandwaspresentinboth3-hourinducedsamples.Sincethebandappearedintheno-insert plasmid,inresponsetoIPTG,itmusthavebeenaproteininvolvedinplasmidoverexpression. 38

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Figure20:SequencealignmentfororiginalpTXB1with mdh-1 longinsert,L4,L410C. Themdh1 geneintheL4andL410clonesweresequenced.TheoriginalpTXB1+ mdh-1 longsequence isinblack,theL4andL410Csequencesareinred.ThesequenceslabeledL4Cindicate thatbothsequencesarethesame.Lowercasesequencesindicatethelocationofthe mdh-1 gene. Upper-casesequencesarethenativepTXB1sequence.Thepolylinkeroftheplasmidvectoris showninblue,withthemutationintheL410Cclonehighlighted.Afterthebluesection,the inteinsequencebegins. 39

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FinalPuricationonChitinColumn Large-scaleinductionswereperformedwithoriginalL4plasmidandtheL410Cmutated plasmid,foranalpurication.Pelletscontaining200mLofcellsfromeachinductionwere resuspendedin5mLcelllysisbuerforpuricationonthechitincolumn.Thiswasmuch lessvolumethanthepreviouspurications,resultinginamuchmoreconcentratedcelllysate solution.Inaddition,morechitinbeadswereusedineachcolumn,toensureadequatebinding surfacefortheMDH-1-intein-CBDexpressionproduct.Theclariedcellextractswereappliedto thecolumnsveryslowly.Eachcolumnwaselutedinvefractions.Allofthesampleswereviewed instainedgelsandwesternblots.Foreachclone,onegellabeled`columnsamples'compared theuninduced,crude,pellet,claried,ow-through,resin,wash,andquickushsamples,and theothergellabeled`elutionfractions'compareduninduced,claried,ow-throughandall elutionsamples.Thewesternblotswerebestforviewingcolumnsamplessincetheytrackthe locationsofchitinbindingdomains.Sincethechitinbindingdomaincleavedooftheprotein withtheineintag,theelutedproteincouldnotbeviewedonthewesternandwasbestvisualized incoomassie-stainedgels. IntheL410CcolumnsamplewesternFigure21,therewasaveryfaintinducedbandat 65kDawhichwasnotdetectableinthestainedgel[notshown].Thisindicatesfairlyweak expression.However,intheRes2lanesecondresinsample,takenaftercleavageandelution ofthesamegure,therewasathickbandatabout30kDa-thisbandrepresentedthecleaved inteintags,containingthechitinbindingdomains,andthusindicatedthatasignicantamount ofproteinboundtoandsubsequentlyelutedfromthecolumn.The65kDabandintheL4 columnsamplewesternFigure23wasalsoveryfaint.Furthermore,theL4Res2sampledoes notindicatesignicantcleavage. IntheL410CelutionsamplegelFigure22,thestrongestMDH-1bandswereat47kDain theE2andE3fractions.WhiletheelutedMDH-1shouldbeslightlysmallerthanthisabout 35kDa,itisstillreasonabletoassumethatthesebandsrepresentedthedesiredprotein.The same47kDabandwaspresentinthetheL4non-mutatedelutionfractionsFigure24,but wasalmostimperceptiblyfaint.Thisreects,expectedly,poorcleavageeciencyduetothe serinewhichwasadjacenttotheinteinsequenceintheL4clone. Theseresultssuggestthatthesite-directedmutagenesisofthe mdh-1 -inteininterfacewas necessaryinorderforthepuricationschemetobesuccessful. 40

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Figure21:L410CChitinColumnPuricationSamplesWesternblot Toparrowindicatesfaint inducedMDH-1bandat65kDa.Bottomarrowindicatesthecleavedintein/CBDtagsbound tothecolumnresinafterelution.Unin=IPTGwasnotintroducedtothesecultures.Cru= Totalinducedcellcultures,lysedbysonication.Pel,Clar=Pellet/Supernatantrespectively afterinducedcellsampleswerelysedandcentrifugedtopelletunwantedcellcomponents.Ladder measuredinkDa.FT=Columnow-throughduringapplicationoftheclariedlysatetothe chitincolumn.Res1=Chitinresinsamplesaftercelllysatewasloadedontothecolumn.Wash =washsample,afterloadingcelllysate.QF=Flow-throughduringquickapplicationofDTTcontainingelutionbuer.Res2=Resinsampleafterelution. 41

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Figure22:L410CElutionfractionsCoomassie-stainedgel Successfulchitincolumnpurication; ArrowindicateselutedMDH-1bands.Unin=IPTGwasnotintroducedtothesecultures.Clar =Supernatantafterinducedcellsampleswerelysedandcentrifugedtopelletunwantedcell components.Flo=columnow-throughduringapplicationoftheclariedlysatetothechitin column.E1-E5=columnelutionsamples. 42

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Figure23:L4notmutatedChitinColumnPuricationSamplesWesternblot. ArrowindicatesfaintinducedMDH-1bandat65kDa.Unin=IPTGwasnotintroducedtothesecultures. Cru=Totalinducedcellcultures,lysedbysonication.Pel,Clar=Pellet/Supernatantrespectivelyafterinducedcellsampleswerelysedandcentrifugedtopelletunwantedcellcomponents. LaddermeasuredinkDa.FT=Columnow-throughduringapplicationoftheclariedlysate tothechitincolumn.Res1=Chitinresinsamplesaftercelllysatewasloadedontothecolumn. Wash=washsample,afterloadingcelllysate.QF=Flow-throughduringquickapplicationof DTT-containingelutionbuer.Res2=Resinsampleafterelution. 43

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Figure24:L4notmutatedElutionfractionsCoomassie-stainedgel Unsuccessfulchitincolumnpurication,duetopoorcleavageeciency.ArrowindicatesveryfaintelutedMDH-1 bands.Unin=IPTGwasnotintroducedtothesecultures.Clar=Supernatantafterinduced cellsampleswerelysedandcentrifugedtopelletunwantedcellcomponents.Flo=columnowthroughduringapplicationoftheclariedlysatetothechitincolumn.E1-E5=columnelution samples. 44

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EnzymeCharacterization UsingtheL410Celutionfraction#3,assayswereconductedtodeterminetheactivityofthe MDH-1inthesamples.BecauseNADHabsorbslightat340nm,whileNAD + doesnot,the NADH-dependentconversionofoxaloacetateintomalatecanbeeasilyanalyzedbymonitoring thedropinabsorbanceat340nm. Therst0.5mLreactionscontained60 MNADH,150 MOxaloacetate,and20 Lof theE3MDH-1sample.Thereactionwasscannedforabsorbanceat340nmfor4minutes. Thesepreliminaryassaysshowedsignicantactivity,sotheassaysfollowingcontainedonly 10 LofMDH-1sample.AssayswereperformedwithseveralotherconcentrationsofOAA, between0-150 M,eachwith10 LoftheE3MDH-1sample.Thescanproducesagraphof absorbanceat340vs.time.Eachabsorbanceversustimegraphwasttoanexpoentialdecay inOriginOriginLab.Thederivativeofeachtequationwasfound,andtheintitialratet=0 wascalculated.AMichaelis-Mentencurveofoxaloacetateconcentrationversusinitialrateof reactionwasprepared.Theasymptoteofthisexponentialcurveindicatesthemaximumreaction velocity,V max ,whichisthereactionvelocitywhenallenzymeactivesitesaresaturated.The enzymeconcentrationwhichcorrespondsto 1 2 V max ,calledthe K m ,isausefulparameterbecause itshowswhentheenzymeishalf-saturatedwithsubstrate.TheMichaelis-Mentencurveisshown inFigure25.TheV max was0.421Abs/min,andtheK m was26.6 MOAA. UsingBeer'slaw,theV max wasconvertedtomoreusefulunits.Beer'slawstates A = lc whereA=absorptionnounits, =theextinctioncoecientofNADH=6220M )]TJ/F7 6.9738 Tf 6.227 0 Td [(1 cm )]TJ/F7 6.9738 Tf 6.227 0 Td [(1 ,l =thepathlengthofthecuvette=1cm,andc=theNADHconcentration.Since A=l = c 0.421Abs/mincorrespondsto 0 : 421 = M )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 cm )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 : 0 cm =68 M ofNADHconvertedperminute. ThesisstudentSaraBondiconductedsimilarassays,using2 LofMDH-1samplein0.6 mLassays.ShefoundtheV max tobeapproximately0.2Abs/min,correspondingto32 M NADH/min.TheK m inherexperimentwas13 M.Bondi2002 TheMDH-1elutionsamplesLCE1-E4andL4E2-E3weredialyzedwithphosphatebuerin ordertoremovetheDTTinthesamples.TheDTTwasproblematicbecauseitinterfereswith theDCproteinassaysBio-Rad,whichdetermineproteinconcentration.Afterdialysis,the sampleswereanalyzedusingtheDC-proteinassayonamicroplate.Theseassaysareextremely sensitive,andpipettingmustbedoneverycarefully.Usingthesamplesofknownconcentration, preparedwithbovineserumalbumin,theGen5softwarerstdeterminesastandardcurveof 45

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Figure25:Michaelis-MentenCurve,[OAA]vs.V 0 Proteinactivityassayswereperformed using60 MNADHandvaryingconcentrationsofOxaloacetatebetween0-150 M.Each0.5 mLassay,conductedatroomtemp,used10 LofMDH-1sampleL410CfractionE3.The assayswerescannedat340nmfor4minutes,andinitialreactionratesV 0 werefoundfor eachOAAconcentration. concentrationvs.absorptionat750nm.ThisstandardcurveisshowninFigure26. Thesoftwarethendeterminestheconcentrationoftheunknownsamplesbycomparingthe A 750 ofeachsampletothestandardcurve.Overall,theconcentrationsofeachfractiondid notcorrespondwiththeintensityoftheelutionbandsinFigure22.InFigure22,E2andE3 appeartobethemostconcentratedfractions.However,intheproteinassays,fractions1,2and 4appearedtobemoreconcentratedthanfraction3Table5.Thesediscrepanciescouldbe duetoresidualDTTinthesamples.Table5showsthevaluesobtainedfortheL410Celution fractions.Onlyfraction3wasusedforenzymeassays. Table5:ProteinConcentrationofL410CE3ElutionFractions. Proteinconcentrationwas measuredbyaDCproteinassayBio-Rad.Thisassayproducesacoloredproductproportional totheproteinconcentrationinthesample.Concentrationsaremeasuredbytheabsorbanceat750 nmandcomparedtothestandardcurveinFigure26.Thereadercannotdetectconcentrations higherthan1565 g/mL. Sample Concentration g/mL Average Std.Dev. L410CE1 1050.88 1001.50 1025.97 35.2 L410CE2 1399.69 1565 L410CE3 768.51 718.68 743.593 35.2 L410CE4 1565 1565 46

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Figure26:ProteinConcentrationStandardCurve. Sampleswithknownconcentrationsofbovine serumalbuminwerepreparedaspartoftheDCproteinassay,acolorimetricassaytodetermine proteinconcentration.Theabsorptionat750nmwasmeasuredforeach,andagraphprepared ofconcentrationvs.A 750 .Thebluedots/errorbarsindicatethetwoactualreadingsforeach preparedstandardsample,andtheorangedotsaretheaverage. Thespecicactivity,SA,istheactivityoftheenzymepermgperunitoftime,or molesof substrateconsumedpermgtotalenzymeperminute molmg )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 min )]TJ/F7 6.9738 Tf 6.227 0 Td [(1 .Oftenthisvalueis reportedinUnits/mg,where1unit=1 molmin )]TJ/F7 6.9738 Tf 6.227 0 Td [(1 .TheSAvaluewasobtainedbyusingthe equation SA= V max volumeofassay/concentrationofsamplexvolumeofMDH-1sampleused. Usingtheaverageconcentrationvalues,thefollowingvalueswerefoundfortheSpecicActivity ofMDH-1. ML )]TJ/F7 6.9738 Tf 6.227 0 Td [(1 min )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 : 0005 L : 743 mgmL )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 : 010 mL =4 : 6 Unitmg )]TJ/F7 6.9738 Tf 6.226 0 Td [(1 ThisSAvalue,4.6Units/mg,isslightlyhigherthanthevalueof4.1U/mgfoundbythesis studentSaraBondi.Sarapredictedthatimpuritiesinthesamplesmayhaveloweredthespecic activityBondi2002.TheenzymeusedinSara'sprojectwasfusedtoaHis-tagwhichmayhave slightlyreducedtheactivity.K m andSAvaluesformMDHinavarietyoforganismsisshown inTable6. 47

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Table6:ComparisonofK m andSpecicActivityValuesformMDHinVariousOrganisms.C. elegans MDH-1apparentlyhasaslightlylowerthanaverageK m ,andreasonableactivitycomparedtosimilarorganismsearthworms.MDHsinmicroorganismsgenerallyhavesignicantly higheractivity.Assayconditionsvariedslightlybetweenstudies,butwerebetweenpH7.4-8.5 and20-25 Cexceptfor L.major ,whichwasassayedat37 C.ForEukaryoticorganisms, valuescorrespondtomitochondrialMDH,except P.falciparum PfMDHiscytosolic;amMDH isoformmaynotexistinthisorganism.Abouttheseorganisms: P.sansibaricus ,epigeicabove surfaceearthworm. M.posthuma, endogenictopsoil-dwellingearthworm. L.mauritii, anecic deepburrowingearthworm. C.batrachus, Freshwatercatsh. L.major, parasiticprotozoa. E. granulosus, parasitictapeworm. P.infestans, potatoblightwatermold. P.falciparum, parasitic protozoamalaria. C.glutamicum, gram-positivesoilbacteria. P.stutzeri, gram-negativesoil bacteria. Organism K m SpecicActivity MOAA Units/min Reference EukaryoticSpecies |{ |{ |{ C.elegans 26.6 4.6 P.sansibaricus { 7 Tripathietal.2009 L.mauritii { 6 Tripathietal.2009 M.posthuma { 0.4 Tripathietal.2009 C.batrachus 125 0.41 Mishra&Shukla1997 L.major 56 932 Lerouxetal.2006 E.granulosus 4 238 Agueroetal.2004 P.infestans 22 1166 Lopez-Calcagnoetal.2009 P.falciparumPfMDH 50 3.9 Pradhanetal.2009 BacterialSpecies |{ |{ |{ C.glutamicum 57 517 Gendaetal.2003 P.stutzeri 32 2460 Labrou&Clonis1997 48

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Conclusions ThisprojectusedtheIMPACTsystemNEBtoexpressandpurify C.elegans MalateDehydrogenaseMDH-1.Thesystemincludedanintein-chitinbindingdomainCBDtag,which isaninterestingalternativetoamoretraditionalHis-tag.Theinduciblecleavageoftheinteinis certainlythemostattractivefeatureofthesystem,asitallowsforeventualelutionofthenativestateproteinincontrasttoaHis-tagwhichremainsontheproteinafterpurication,possibly hinderingitsactivity.Theinteinfeatureprovedtobemorecomplicatedthanitseemed,asthe targetgene-inteintaginterfaceneededtoberedesignedwithintheplasmidDNA.Originally, theplasmidsequencewasdesignedtohaveaSerresidueadjacenttotheintein,butthisamino acidisnotconducivetointeincleavage.Site-directedmutagenesiswasperformedinorderto alterthesequence,replacetheaminoacidandensureadequatecleavageoftheMDH-1-intein precursoronceitwasboundtothecolumn. Intheend,theintein-CBDtagwashighlysuccessful.TheCBDoeredhighlyspecicbinding,allowingforverythoroughwashingofthecolumnandsubsequentlyverylittlecontamination oftheproductasevidencedbygels/westernblotsoftheelutionfractions.Theentirepuricationschemewasperformedonasinglecolumn,whichminimizedproductlossandalsoreduced thetimenecessarytoperformthepurication. Inpreviousexperiments,theenzymeswentthroughoneortworoundsofdialysisat4 C beforekineticanalysis.Keepingtheproteinsatsuchhightemperaturesforextendedperiods oftimemaylowertheiractivity.Inthisexperiment,thekineticassayswereperformedbefore dialysis.ThedialysiswasperformedlaterinordertoremovetheDTTintheproteinsamples sothatconcentrationscouldbemeasured.Theproteinsampleswerequitefreshforthekinetic assayswhichlikelyhelpedimprovetheoverallactivity.TheV max ofMDH-1wasfoundtobe 0.421Abs/min,or68 MofNADHconsumed/min.TheK m wasfoundtobe26.6 MOAA. Unfortunately,concentrationassaysproducedslightlyunreliableresults,duetodicultpipetting andpossiblyresidualDTT.Thecalculationofspecicactivityoftheenzymedependsonthese measurements,andthusinfutureexperimentsthesamplesshouldbedialyzedinlargervolumes ofbuer,and/orforlongerperiodsoftime. 49

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Thespecicactivitywascalculatedtobe4.6Units/mg.Thisisslightlyhigherbutfairly consistentwiththevalueof4.1U/minfoundbythesisstudentSaraBondiBondi2002.ComparingtheseresultswithkineticparametersfoundforotherorganismsTable6,itseemsthat C.elegans' MDH-1K m valueisslightlylowerthanaverage.MDH-1specicactivityisrather averagewhencomparingtoorganismssuchascatsh C.batrachus orearthworms P.sansibaricus,L.mauritii,M.posthuma .Otherorganisms,includingsoilbacteriaandavarietyof parasiticorganisms,seemtohaveMDHisoformswithsignicantlyhigheractivity.Interestingly, thecytoplasmicPfMDHofthemalaria-causingprotozoa P.falciparium hadakineticprole mostsimilartotheMDH-1inthecurrentstudy. ExperimentsinthefuturewouldbenetfromoverallimprovementinproteinMDH-1expression.Althoughthemodestexpressionobservedinthisprojectwasmorethanadequate,it wasdiculttodetectusingSDS-PAGEandwesternblots.Futurestudentscouldfocusonmore kineticexperiments,possiblyexamininghowMDH-1activityvarieswithpHortemperature. Structureelucidationexperimentscouldalsobeperformed. 50

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SolutionsAppendix Electrophoresissolutions 5XSDStankbuer 7.56gtrisbase 500mL36gglycine 2.5gSDS DIwaterto500mL 10%acrylamideresolvinggel 1.5mL40%acrylamide 3.0mL2Xrunninggelbuer 1.5mLnanopurewater *Topolymerizegel,add35 L10%APSand15 LTEMEDbeforepouring.Afterpouring,cover withabout1mL0.1%SDSsolution. Stackinggel 0.38mL40%acrylamide 0.75mL4Xstackinggelbuer 1.9mLnanopurewater *Topolymerizegel,add35 L10%APSand15 LTEMEDbeforepouring.Afterpouring,add plasticcombtoformwells. 10%acrylamideureagel 3.15gurea 1.88mL40%acrylamide 0.375mL20XSBbuer addnanopurewaterto7.5mL *Topoylmerizegel,add35 L10%APSand15 L TEMEDbeforepouring. 2Xurealoadingbuer 5gurea 2mL5XTBE afewmgbromophenolblue nanopurewaterto10mL GelElutionbuer 3mL0.3MNaOAc,pH5.2 30mL3mL1%SDS 60 L0.5MEDTA nanopurewaterto30mL ltersterilize 1%agarosegel 35mL1XSBbuer 0.35gagarose Microwave,stirtodissolveagarose Add3.5 LGelRedbeforepouring 51

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WesternBlotsolutions TransferBuer 600mLMeOH 3L9.07gTrisbase 43.2gGlycine DIwaterto3L WesternBlotAPbuer 4mL1MTris,pH9.5 40ml0.8mL5MNaCl 1mL200mMMgCl 2 nanopurewaterto40mL Rightbeforeusing,addlightsensitive: 264 LNBT 132 LBCIP Tris-BueredSalineTween-20 5mL1MTris,pH7.9 TBST15mL5MNaCl 500mL250 LTween-20 nanopurewaterto500mL IMPACTpuricationcolumnsolutions ColumnBuer 10mL1Mtris-HClpH8.0mM 500mL50mL5MNaClmM Bringto500mLwithnanopurewater CellLysisbuer 1mL25xcOmpleteRocheAppliedSciencesproteaseinhibitorcocktail 25mL25 Ltween-20.1% Bringto25mLwithcolumnbuer ElutionBuer 1mL1MstockDTTmM 20mLColumnbuerto20mL StrippingSolution 0.3MNaOH Enzymekineticssolutions KineticAssayBuer 10mL1MPotassiumphosphate,pH7.5.1M 100mL0.75gKCl.1M nanopurewaterto100mL Dialysis/Storagebuer 150mLglycerol% 500mL3.73gKCl.1M 15mL1MPotassiumphosphate,pH7.5mM 52

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Figure27:QIAprepSpinMiniprepKitProtocol,UsingaMicrocentrifuge.Thisprotocolis designedforthepuricationofupto20 ghigh-copyplasmidDNAfrom15mlovernight E.coli cultureinLBmedium. ModiedgurefromtheQIAprepMiniprepHandbook,SecondEdition, December2006.QIAGEN,Allrightsreserved. 53

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References Agueroetal.Purication,cloning,andexpressionofthemitochondrialmalatedehydrogenase mMDHfromprotoscolicesof Echinococcusgranulosus. Molecular&BiochemicalParasitology. 137,207-214. Amarneh,BilalandStevenB.Vik.DirectTransferofNADHFromMalateDehydrogenaseto ComplexIin Escherichiacoli. CellBiochemistryandBiophysics. 42. Bell,Jessica,etal.StructuralAnalysesofaMalateDehydrogenasewithaVariableActive Site." JournalofBiologicalChemistry. 276:33,31156-31162. Biochemistry ,2ndedition.Garrett,ReginaldH.andCharlesM.Grisham.Brooks/Cole CendageLearning,Florence,KY. Biochemistry ,6thedition.Berg,Jeremyetal.W.H.FreemanandCompany,NewYork, NY. Bondi,Sara.B.A.thesis,NewCollegeofFL.Sarasota,FL,2002. Birktoft,JensJ.andLeonardJ.Banaszak.ThePresenceofaHistidine-AsparticAcidPaiirntheActiveSiteof2-HydroxyacidDehydrogenases." JournalofBiologicalChemistry. 258:1,452-482. Gendaetal.PuricationandCharacterizationofMalateDehydrogenasefrom Corynebacterium glutamicum. JournalofBioscienceandBioengineering. 95:6,562-566. Gieti,Christine.PartitioningofMalateDehydrogenaseIsoenzymesintoGlyoxysomes,Mitochondria,andChloroplasts." PlantPhysiol. 100,557-559. Goward,C.andD.Nichols.Malatedehydrogenase:Amodelforstructure,evolution,and catalysis." ProteinScience. 3,1883-1888. Hall,M.D.,andL.J.Banaszak.CrystalStructureofaTernaryComplexof Escherichiacoli MalateDehydrogenaseCitrateandNADat1.9AngstromResolution" J.Mol.Biol. 232,213-222. Holt,SuzanJ.,andDonaldL.Riddle.SAGEsurveys C.elegans carbohydratemetabolism: evidenceforananaerobicshiftinthelong-liveddauerlarva." MechanismsofAgeingand Development. 124,789-800. Kondrashov,FyodorA.,etal.EvolutionofglyoxylatecycleenzymesinMetazoa:evidenceof multiplehorizontaltransfereventsandpseudogeneformation." BiologyDirect. 1:31. LabrouandClonis.L-MalateDehydrogenasefrom Pseudomonasstutzeri: Puricationand Characterization." ArchivesofBiochemistryandBiophysics. 337:1,103-114. 54

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Lerouxetal.FunctionalcharacterizationandsubcellularlocalizationofthethreemalatedehydrogenaseisozymesinLeishmaniaspp." Molecular&BiochemicalParasitology. 149,7485. Lopez-Calcagnoetal.Cloning,expressionandbiochemicalcharacterizationofmitochondrial andcytosolicmalatedehydrogenasefrom Phytophthorainfestans ." MycologicalResearch. 113,771-781. Minarik,etal.MalateDehydrogenases{StructureandFunction." Gen.Physiol.Biophys. 21,257-265. Mishra&Shukla.ImpactofEndosulfanonCytoplasmicandMitochondrialLiverMalateDehydrogenasefromtheFreshwaterCatsh Clariusbatrachus ." Comp.Biochem.Physiol. 117C:1,7-18. Molenaar,Douwe,etal.FunctionsoftheMembrane-AssociatedandCytoplasmicMalateDehydrogenasesintheCitricAcidCycleof Corynebacteriumglutamicum. JournalofBacteriology. 182:24,6884-6891. Morgunov,IgorandPaulA.Srere.InteractionbetweenCitrateSynthaseandMalateDehydrogenase;SubstrateChannelingofOxaloacetate." JournalofBiologicalChemistry. 273:45,29540-29544. Pradhanetal.Structureandfunctionof Plasmodiumfalciparum malatedehydrogenase:Role ofcriticalaminoacidsinco-substratebindingpocket." Biochimie. 91,1509-1517. Shanks,Christopher.B.A.thesis,NewCollegeofFL.Sarasota,FL,2008. Tomitaetal.Structuralbasisforthealterationofcoenzymespecicityinamalatedehydrogenasemutant." BiochemicalandBiophysicalResearchCommunications. 347, 502-508. Tripathietal.Impactofphorateonmalatedehydrogenases,lactatedehydrogenaseandproteinsofepigeic,anecicandendogeicearthworms." PesticideBiochemistryandPhysiology. 95,100-105. VanDerRest,MichelE.,etal.FunctionsoftheMembrane-AssociatedandCytoplasmicMalate DehydrogenasesintheCitricAcidCycleofEscherichiacoli." JournalofBacteriology. 182:24,6892-6899. 55


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