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JournalofVisualizedExperiments www.jove.com VideoArticle A Protocol for the Production of KLRG1 Tetramer StephanieC. Terrizzi,Cindy Banh,Laurent Brossay DepartmentofMolecularMicrobiologyandImmunology,BrownUniversity Correspondenceto:[email protected] URL:http://www.jove.com/index/Details.stp?ID=1701 DOI:10.3791/1701 Citation:Terrizzi S.C.,Banh C.,Brossay L.(2010).AProtocolfortheProductionofKLRG1Tetramer.JoVE.35.http://www.jove.com/index/Details.stp?ID=1701,doi:10.3791/1701 Abstract Killercelllectin-likereceptorG1(KLRG1)isatypeIItransmembraneglycoproteininhibitoryreceptorbelongingtotheCtypelectin-likesuperfamily. KLRG1existsbothasamonomerandasadisulfide-linkedhomodimer.Thiswell-conservedreceptorisfoundonthemostmatureandrecently activatedNKcellsaswellasonasubsetofeffector/memoryTcells. UsingKLRG1tetrameraswellasothermethods,E-,N-,andR-cadherinswereidentifiedasKLRG1ligands.TheseCa2+-dependentcell-cell adhesionmoleculescomprisesofanextracellulardomaincontainingfivecadherinrepeatsresponsibleforcell-cellinteractions,atransmembrane domainandacytoplasmicdomainthatislinkedtotheactincytoskeleton. GenerationoftheKLRG1tetramerwasessentialtotheidentificationoftheKLRG1ligands.KLRG1tetramerisalsoauniquetooltoelucidatethe rolescadherinandKLRG1playinregulatingtheimmuneresponseandtissueintegrity. Protocol Preparation of Inclusion Bodies 1. Inoculate4x500mlflasksofTBeachcontaining50μg/mlofcarbenicillinandchloramphenicolwithanovernightcultureofbacteria expressingtheKLRG1plasmidconstruct.WhentheODreaches0.6Åat600nm,inducewiththeadditionof0.4MIPTGandincubatethe cultureforanadditional4hoursshakingat37°C. 2. ResuspendtheharvestedcellsinresuspensionbufferpH=8.0(50mMTris-HCl,25%(W/V)sucrose,1mMEDTA,10mMDTT).Note:pellets canbefrozenatthisstage. 3. Poolnomorethan60mlofbacterialresuspensionsinapolypropylenebeaker.Ifnecessaryadjustto60mlwithTEbufferpH8.0andstir mixtureathalfspeed. 4. Tostirringmixtureadddropwise:lysozyme(final=1mg/ml),MgCl2(final=5mM),2mgDNAseIin50%glycerolcontained75mMNaCl, Triton-X100(final=1%),DTT(final=10mM). 5. Sonicatethesolutiononicefor1.5minandcentrifugethelysatesat5,000RPMfor10minat4°C. 1. Decantthesupernatant. 2. Add15mlofwashbufferpH=8.0(50mMTris-HCl,0.5%TritonX-100,100mMNaCl,1mMEDTA,1mMDTT). 3. Onice,sonicatesolutionfor1.5minuntilthepelletiscompletelyresuspended. 4. Centrifugethesamplesat5,000RPMfor10minat4°C. 5. Repeatthewash5times. 6. Repeat5bwithwashbufferwithoutTritonX-100,centrifugeandresuspendin4mlofTEBuffer. 7. TakethewetweightinclusionbodiesslurryandstoreinTEbufferataconcentrationof30to100mg/ml. Refolding of KLRG1 1. Make1LofrefoldingbufferpH=8.0(0.4MArginine-HCl,0.1MTrispH8-8.3,2mMEDTA,0.2mMPMSF,3EDTA-freeproteaseinhibitor tablets,5mMGSHand0.5mMGSSG)andchillto4°Cwhilestirring. 2. Preparetheinclusionbodiesforinjectionintothefoldingmixture:Itisidealtomake510injectionseachwith0.250.5μMconcentrationso thatthefinalconcentrationoftheproteinwillbe23μM. 1. Meltthevolumeof50mgofinclusionbodiesslurryin7MGnHClwith10mMβ-mercaptoethanol. 2. Keeptheinclusionbodies/GnHClmixtureat37°Cfor30-40minandvortexevery5mintoensurecompletemelting(slurrywillbecome clearwhencompletelymelted). 3. Centrifugeatmaxspeedfor30mininmicrocentrifugeat4°Candtransferthesupernatanttoa15mlcentrifugetube.Donottotransfer anyofthesmallblackishpellet. 3. Adjustvolumewithinjectionbuffer(3MGnHCl,10mMNaAcetate,pH4.2,10mMEDTA). 4. Inject1mlofdilutedinclusionbodieseveryhour.Wheninjecting,stirathighspeed,add1mlofdilutedinclusionbodiesdropbydropwith2 secondsbetweeneachdrop.Wheninjectionisdone,stiratlowspeed. 5. Continueovernightstirringatlowspeedat4°C. Concentration of Refolding Reactions 1. FollowingMilliporesprotocol,concentratethe1Lofpre-filtered(0.22μmfiltered)refoldingreactionusinganAmiconStirredCellandYM10 NMWL10Kultrafiltrationmembrane(Millipore)to~10ml. 2. Concentrateto≤2mlusingaCentriplusYM-1010KMWCO(Millipore). Copyright©2010JournalofVisualizedExperiments Page1of2 JournalofVisualizedExperiments www.jove.com Purification of KLRG1 tetramer 1. SetuptheAKTAFPLCat4°CaccordingtoGEHealthcaremanuals. 2. PurifythemonomerformofKLRG1bysizeexclusionchromatographyusingaSephacrylS-20016/60high-resolutioncolumn(GE Healthcare)in20mMHEPESand150mMNaCl.(SeeFigure1). 3. Concentrateto1mlusingCentriplusYM-1010KMWCO(Millipore). 4. UseBirAenzymeaccordingtoAviditysprotocoltobiotinylatetheKLRG1monomer. 5. Thefreebiotiniseliminatedbysizeexclusionchromatographyasin2. 6. KLRG1moleculeistetramerizedbymixingKLRG1monomerwith4foldmolarexcessPEconjugatedstreptavidin(BDBiosciences). Representative Results Figure1.RepresentativepositiveresultsfromtheAKTAFPLCsizeexclusionchromatographyoftherefoldedKLRG1.Thegraphshowsthe separationofthemonomer(83.52ml),dimer(56.36ml),andmultimer(36.26ml)formoftherefoldedKLRG1.Thepeakat94.25mlrepresentsthe bufferexchange. Discussion Inthisprotocol,itisshownhowtopurify,biotinylate,andtetramerizeKLRG1.KLRG1tetramercanbeusedtolabelKLRG1ligandsbyflow cytometry.Althoughrefoldingconditionswillhavetobetestedempiricallyforeachprotein,otherC-typelectinscouldbetetramerizedusinga similarprotocol.Usingtetramerizedproteinsasprobes,ligandstoorphanC-typelectinreceptorscouldpotentiallybeidentified. Acknowledgements WethankDr.Naidenkoforhelpinoptimizingtherefoldingconditions.ThisworkwassupportedbyNIHresearchgrantAI58181toLaurent Brossay.CindyBanhissupportedbyNIHNRSAF31AI080230. StephanieTerrizziandCindyBanhcontributeequallytothiswork. References 1. Tessmer,M.S.,Fugere,C.,Stevenaert,F.,Naidenko,O.V.,Chong,H.J.,Leclercq,G.&Brossay,L.KLRG1bindscadherinsandpreferentially associateswithSHIP-1.IntImmunol19,391-400(2007). 2. Grundemann,C.,Bauer,M.,Schweier,O.,vonOppen,N.,Lassing,U.,Saudan,P.,Becker,K.F.,Karp,K.,Hanke,T.,Bachmann,M.F.& Pircher,H.Cuttingedge:identificationofE-cadherinasaligandforthemurinekillercelllectin-likereceptorG1.JImmunol176,1311-1315 (2006). 3. Ito,M.,Maruyama,T.,Saito,N.,Koganei,S.,Yamamoto,K.&Matsumoto,N.Killercelllectin-likereceptorG1bindsthreemembersofthe classicalcadherinfamilytoinhibitNKcellcytotoxicity.JExpMed203,289-295(2006). 4. Li,Y.,Hofmann,M.,Wang,Q.,Teng,L.,Chlewicki,L.K.,Pircher,H.&Mariuzza,R.A.StructureofnaturalkillercellreceptorKLRG1boundto E-cadherinrevealsbasisforMHC-independentmissingselfrecognition.Immunity31,35-46(2009). 5. Nakamura,S.,Kuroki,K.,Ohki,I.,Sasaki,K.,Kajikawa,M.,Maruyama,T.,Ito,M.,Kameda,Y.,Ikura,M.,Yamamoto,K.,Matsumoto,N.& Maenaka,K.MolecularbasisforE-cadherinrecognitionbykillercelllectin-likereceptorG1(KLRG1).JBiolChem,inpress(2009). Copyright©2010JournalofVisualizedExperiments Page2of2