Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates 英文参考文献.docVIP

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Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates 英文参考文献.doc

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Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates 英文参考文献

EnzymaticAnalysisofRecombinantJapanese EncephalitisVirusNS2B(H)-NS3proProteasewith FluorogenicModelPeptideSubstrates MuhammadJunaid1,2,ChakardChalayut1,AnnaSehgelmebleTorrejon2,ChananAngsuthanasombat1, IrynaShutava2,MarisLapins2,JarlE.S.Wikberg2,GerdKatzenmeier1* 1Laboratory of Molecular and Cellular Microbiology, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakornpathom, Thailand, 2Department of PharmaceuticalBiosciences,DivisionofPharmacology,UppsalaUniversity,Uppsala,Sweden Abstract Background:Japaneseencephalitisvirus(JEV),amemberoftheFlaviviridaefamily,causesaround68,000encephalitiscases annually,ofwhich20–30%arefatal,while30–50%oftherecoveredcasesdevelopsevereneurologicalsequelae.Specific antiviralsforJEVwouldbeofgreatimportance,particularlyinthosecaseswheretheinfectionhasbecomepersistent.Being indispensableforflaviviralreplication,theNS2B-NS3proteaseis apromising targetfordesignofanti-flaviviralinhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemicalcharacterizationofJEVNS2B(H)-NS3proprotease. Methodology/PrincipalFindings:Thefull-lengthsequenceofJEVNS2B-NS3genotypeIIIstrainJaOArS982wasobtainedas a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and clonedintothepTrcHisAvector.Hexahistidine-taggedNS2B(H)-NS3pro,expressedinE.coliassolubleprotein,waspurified to.95%puritybyasingle-stepimmobilizedmetalaffinitychromatography.SDSandimmunoblottingofthepurified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10kDabands,respectively.Kineticparameters,Kmandkcat,forfluorogenicproteasemodelsubstrates,Boc-GRR-amc,Boc- LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect cor