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1、RenillaLuciferaseActivities1IvoQueIntroductionFireflyandRenillaluciferases,becauseoftheirdistinctevolutionaryorigins,havedissimilarenzymestructuresandsubstraterequirements.Thesedifferencesmakeitpossibletoselectivelydiscriminatebetweentheirrespectivebioluminescentreactions
2、.Fireflyluciferaseisa61kDamonomericproteinthatdoesnotrequirepost-translationalprocessingforenzymaticactivity.Thus,itfunctionsasageneticreporterimmediatelyupontranslation.Photonemissionat560nmisachievedthroughoxidationofbeetle2+luciferininareactionthatrequiresATP,MgandO2(F
3、igure1).Underconventionalreactionconditions,theoxidationoccursthroughaluciferyl-AMPintermediatethatturnsoververyslowly.Asaresult,thisassaychemistrygeneratesa“flash”oflightthatrapidlydecaysafterthesubstrateandenzymearemixed.Renillaluciferase,a36kDamonomericprotein,mayfunct
4、ionasageneticreporterimmediatelyfollowingtranslation.TheluminescentreactioncatalyzedbyRenillaluciferaseutilizesO2andcoelenterateluciferin(coelenterazine)(Figure1).Intheassay,thekineticsoftheRenillaluciferasereactionprovidesastabilizedluminescentsignalthatdecaysslowlyovert
5、hecourseofthemeasurement.1LeidenUniversityMedicalCentre,DepartmentEndocrinology,TheNetherlands;IQue@lumc.nlApplicationNoteRenillaluciferasehasaspectralpeakat480nmandissodiumdependentandATPindependent.ExperimentBhaumikandGambhirhaverecentlydemonstratedthatRenillaluciferase
6、(Rluc)isapromisingbioluminescencereportergenethatcanbeusedfornoninvasiveopticalimagingofreportergeneexpressioninlivingmice,withtheaidofacooledchargedcoupledevice(CCD)camera.Inourstudy,weexploretheexpressionofanovelEnduRenandViviRenRenillaluciferasereportergene(Promega)inv
7、itroandinvivo.B02F11-N11cancercellsweretransfectedwithpCMV-lucand/orpCMVRlucplasmid.72hoursaftertransfectionandtrypsinizationthecellswerewashedinPBS.10E4cellswereusedfortheinvitroexperiment(fig.2).B02F11RL-LucN11(invitro)[substrate]80µM30min8µM0,8µM80µM24h8µM0,8µMImagecou
8、rtesyI.Que,N.V.Henriquez&G.vanderPluijmFigure2.Lightproductionof10E4B02F11RL-LucN11cancercellsex
