体外重组人细胞色素P4502C9酿酒酵母表达体系的构建及

1942009643ChinMedBiotechnol,June2009,Vol.4,No.3DOI:10.3969/cmba.j.issn.1673-713X.2009.03.007··P4502C9P4502C9CYP2C9SaccharomycescerevisiaeCYP2C9CYP2C9*1PCRCYP2C9*2R144CCYP2C9*3I359L3CYP2C9HPLCCYP2C9*1PrismDemoKmBOMCC3KmVmax53CYP2C9*1Km5.34±0.96µmol/LBOMCCCYP2C9*1CYP2C9*2Km16.94±4.7834.73±5.51µmol/LVmax0.21±0.100.12±0.01pmol/min·pmolP4500.012±0.0030.003±0.0001µl/min·pmolP450CYP2C9*35CYP2C9*1CYP2C9*2CYP2C9CYP2C9*2CYP2C9*3CYP2C9P450(3):194-199P4502C9CYP2C9CYP3A4P45020%100CYP2C910%~20%[1]CYP2C932[2]CYP2C9*1CYP2C9*2R144CCYP2C9*3I359LCYP2C9[3-6]CYP2C9TOP10TaqDNApYES2/CTV5-V5BOMCCInvitrogenDNATaKaRaT4PromegaPVDFOSMONI[e-mail]2009-02-202009643ChinMedBiotechnol,June2009,Vol.4,No.3195PIERCEBradfordSigma6-6-NADPBD96CostarGeminiXPS/EMMolecularDevicesUV2550Agilent1200CYP2C9*2CYP2C9*3CYP2C9*1PCRPrimer5CYP2C9*2R144C430bpCTP15’GGAAGAGGAGCATTGAGGACTGTGTTCAAGAGGAAGCCCGC3’P25’GCGGGCTTCCTCTTGAACACAGTCCTCAATGCTCCTCTTCC3’CYP2C9*3I359L1075bpACP15’TGCACGAGGTCCAGAGATACCTTGACCTTCTCCCCACCAGC3’P25’GCTGGTGGGGAGAAGGTCAAGGTATCTCTGGACCTCGTGCA3’PCR2TaqDNA25µlDNA1ng20µmol/L1µl50µlPCR945min9430s5760s7260s357210minPCRKpnXhoT4pYES2/CTTOP10CYP2C9CYP2C9*1CYP2C9*2CYP2C9*3P450SDS-PAGEPVDF4-V5HRPIgGECLCYP2C9350mlSD2%30500mlSD7~8h700mlSG2%36~42h10%0.1mol/LpH7.40.1mmol/LDTT0.1mmol/LEDTA1mmol/LPMSF0.5mm1000×g10min5000×g1h–80CYP2C902.5510204080µmol/LCYP2C9*10.5mg/ml27HPLC-280nm4.6nm×250mmC18A20%pH3~4B30%B20min100%B0.8ml/minPrismDemoKmCYP2C9BOMCC960.5mg/ml10µmol/L5µl20×co-factor66mmol/LNADP26mmol/L6-=110.33µl1mol/LMgCl25µl1mol/LpH7.41µl40U/ml6-0.2µl5mmol/LBOMCC10µl10×10µl100µl963710min409nm460nmkinetics2min160minBOMCC0510204080µmol/L1962009643ChinMedBiotechnol,June2009,Vol.4,No.3PrismDemoKmVmax51112864321894.52.251.1250.5630.2810.140µmol/L3730min50µl100%10µlendpoint18IC50P0.051CYP2C9*2CYP2C9*3ABCYP2C9*2CYP2C9*3ACYP2C9*2BCYP2C9*3Figure1ThesequencesofCYP2C9*2andCYP2C9*3.A:CYP2C9*2;B:CYP2C9*3.550002Km5.34±0.96µmol/L3BOMCCCYP2C9*2CYP2C9*1M1234583000670004750032500M123CYP2C9*14CYP2C9*25CYP2C9*3M:Marker;1:Positivecontrol;2:Negativecontrol;3:CYP2C9*1;4:CYP2C9*2;5:CYP2C9*3;ThefiguresmarkedbesidethelanesarerelativemolecularweightCYP2C9Figure2WesternblottingshowingrecombinantCYP2C9enzymespmol/min·mgReactionrate[pmol/(min·mg)]252015105020406080minTime(min)CYP2C9Figure3TheenzymeactivitiesforrecombinantCYP2C9enzymesweredetectedbyfluorescenthigh-throughputCYP2C9*399%1CYP2C9*1Km20µmol/LVmax0.26pmol/min·pmolP4500.013µl/min·pmolP450CYP2C9*2KmCYP2C9*11VmaxCYP2C9*1CYP2C9*1CYP2C9*2KmP0.055CYP2C9*1CYP2C9*222CYP2C943CYP2C922009643ChinMedBiotechnol,June2009,Vol.4,No.3197CYP2C9*1CYP2C9*2KmVmaxTable1TheMichaelisconstant(Km)andmaximumenzymaticreactionrates(Vmax)ofCYP2C9*1andCYP2C9*2CYP2C9*1CYP2C9*2Kmµmol/L19.680±0.87034.730±5.510Vmaxpmol/min·pmolP4500.260±0.0400.120±0.010µl/min·pmolP4500.013±0.0020.003±0.0001Intrinsicclearancerate[µl/(min·pmolP450)]5CYP2C9*1CYP2C9*2Table2ThedifferenceofinhibitiondegreeswithfivedrugsonCYP2C9*1andCYP2C9*2DrugsCYP2C9*1CYP2C9*2PioglitazoneKetoconazole++++Fluoxetine+++Sulfaphenazole++++++++Tolbutamide+++IC50100µmol/L+50µmol/LIC50100µmol/L++10µmol/LIC5050µmol/L+++1µmol/LIC5010µmol/L++++IC501µmol/LNotes::Thedrughasnoinhibitiontoenzymes(IC50100µmol/L);+:Standsforweakinhibition(50µmol/LIC50100µmol/L);++:Showsmoderateinhibition(10µmol/LIC5050µmol/L);+++:Repressesstronginhibition(1µmol/LIC5010µmol/L);++++:Showsextremelystronginhibition(IC501µmol/L).CYP2C9KmFDA[9]10Gill[11]CYP2C9*2CYP2C9*3CYP2C9*3CYP2C9*2CYP2C9*2CYP2C9*1KmVmaxCYP2C9*270%CYP2C9CYP2C95CYP2C9CYP2C9CYP2C9CYP2C9CYP2C9*1CYP2C9*2CYP2C9CYP2C9*1CYP2C9*2CYP2C9*1CYP2C9CYP2C9*2CYP3A4[9]CYP2C9CYP3A4CYP2C9*1CYP2C9*2CYP2C9*1CYP2C9*2CYP2C9CYP2C9*1CYP2C9*2CYP2C9*2CYP2C9CYP2C91982009643ChinMedBiotechnol,June2009,Vol.4,No.3CYP2C9[1]KirchheinerJ,BrockmöllerJ.ClinicalconsequencesofcytochromeP4502C9polymorphysms.ClinPharmocalTher,2005,77(1):1-16.[2]HongX,ZhangS,MaoG,etal.CYP2C9*3allelicvariantisassociatedwithmetabolismofirbesartaninChinesepopulation.EurJClinPharmacol,2005,61(9):627-634.[3]NakaiK,HabanoW,NakaiK,etal.EthnicdifferencesinCYP2C9*2(Arg144Cys)andCYP2C9*3(Ile359Leu)genotypesinJapaneseandIsraelipopulations.LifeSci,2005,78(1):107-111[4]ScordoMG,AklilluE,YasarU,etal.GeneticpolymorphismofcytochromeP4502C9inaCaucasianandablackAfricanpopulation.BrJClinPharmacol,2001,52(4):447-450.[5]García-MartínE,MartínezC,LaderoJM,etal.HighfrequencyofmutationsrelatedtoimpairedCYP2C9metabolisminaCaucasianpopulation.EurJClinPharmacol,2001,57(1):47-49.[6]YangJQ,MorinS,VerstuyftC,etal.FrequencyofcytochromeP4502C9allelicvariantsintheChineseandFrenchpopulations.FundamClinPharmacol,2003,17(3):373-376.[7]BDBiosciences.HPLCassaymethod[EB/OL].[2008-08-26].=353[8]CrespiCL,MillerVP,PenmanBW.Microtiterplateassaysforinhibitionofhuman,drug-metabolizingcytochromesP450.AnalBiochem,1997,248(1):188-190.[9]FDA,CDER,CBER.GuidanceforIndustrysafetyDrugI