Rapid Modification of Proteins Using a Rapamycin-Inducible Tobacco Etch Virus Protease System 英文参考文献.docVIP

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Rapid Modification of Proteins Using a Rapamycin-Inducible Tobacco Etch Virus Protease System 英文参考文献.doc

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Rapid Modification of Proteins Using a Rapamycin-Inducible Tobacco Etch Virus Protease System 英文参考文献

RapidModificationofProteinsUsingaRapamycin- InducibleTobaccoEtchVirusProteaseSystem DamianJ.Williams,HenryL.Puhl,III,StephenR.Ikeda* LaboratoryofMolecularPhysiology,NationalInstituteonAlcoholAbuseandAlcoholism,NationalInstitutesofHealth,Bethesda,Maryland,UnitedStatesofAmerica Abstract Background: The ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedicalresearch.Specificproteasesprovideapotentialmethodtoselectivelycleaveachosenprotein,butrapidcontrol ofproteaseactivityisdifficult. Methodology/Principal Findings: A heterologous expression system for rapid target-directed proteolysis in mammalian cells wasdeveloped. Thesystem consists of aninducibleNIa proteasefromthetobaccoetch virus (TEVp) andachosen proteinintowhichaTEVpsubstraterecognitionsequence(TRS)hasbeeninserted.Inducibleactivitywasconferredtothe TEVpusingrapamycin-controlledTEVpfragmentcomplementation.TEVpactivitywasassayedusingaFRET-basedreporter construct.TEVpexpressionwaswelltoleratedbymammaliancellsandcompletecleavageofthesubstratewaspossible. Cleavage at 37uC proceeded exponentially with a time constant of approximately 150 minutes. Attempts to improve cleavageefficiencywerehamperedbysubstantialbackgroundactivity,whichwasattributedtoinherentaffinitybetween the TEVp fragments. A second TEVp assay, based on changes in inactivation of a modified KV3.4 channel, showed that functionalpropertiesofachannelcanbeusingalteredusinganinducibleTEVpsystem.Similarlevelsofbackgroundactivity andvariabilitywereobservedinbothelectrophysiologicalandFRETassays. Conclusions/Significance:TheresultssuggestedthatanoptimumlevelofTEVpexpressionleadingtosufficientinducible activity(withminimalbackgroundactivity)existsbutthevariabilityinexpressionlevelsbetweencellsmakesthepresent systemratherimpracticalforsinglecellexperiments.Thesystemislikelytobemoresuitableforexperimentsinwhichthe cell-to-cellvariabilityislessofanissue;forexample,inexperimentsinvolvinglargepopulati