A Protocol for the Production of KLRG1 Tetramer.

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by  Terrizzi, Stephanie C.

JournalofVisualizedExperiments www.jove.com
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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).
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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
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