人FKBP12的基因克隆及其表达产物的生物活性3

ISSN100727626CN1123870öQChin.J.Biochem.Mol.Biol.2000,Vol.16,No.3,322325163200063973(G1998051107)33Tel:[phone],Fax:[phone]E2mail:[e-mail],,19708,,:1999208226,:1999210225FKBP123(,100850)33(,100850)hFKBP12,LRT2PCRpBV220,TJurkathFKBP12L,,pBV220,20%L,,hFKBP12LhFKBP12LFKBP12,,,R392111TheGeneCloningandBioactivityoftheExpressionProductoftheHumanFKBP12PEIWu2hong,HUMei2ru,LIWu2ju,SHENBei2fen(BeijingInstituteofBasicMedicalSciences,Beijing100850,China)LISong(BeijingInstituteofPharmacologyandToxicology,Beijing100850,China)AbstractToobtainbioactivehFKBP12proteinforscreeningnovelneurotrophicdrugs,RT2PCR,computer2aidedgeneexpressiondiscriminatingmodelandM.W.cut2offultrafiltrationwereused.hFKBP12genewasclonedsuccessfullyandthenredesignedoptimallyaccordingtothehighexpressionmodelfortheforeigngene.TheredesignedgenewashighlyexpressedinpBV220vec2torasinclusionbodies,whichwererenaturedandpurifiedtoonlyonebandonSDS2PAGE.ThepurifiedhFKBP12showedpeptidyl2prolylcis2transisomerase(PPIase)activity.ItindicatedthatrecombinanthFKBP12wasbioactivejustasitswildtype’s.KeywordshFKBP12,Geneclone,Procaryoticexpression,Peptidyl2prolylcis2transisomeraseFKBP12FK506(FK5062bindingprotein,FKBP),12kD,14kDL1989,L1994,FKBP1250,FK506[1]LFKBP,FKBP,FK506,,L1997,,[2]LFKBP12,,FKBP12,,L1111pBV220;JM109;EcoRÉBamHÉT4DNAPromega;;N2suc2cinyl2Ala2Ala2Pro2Phe2Q2nitroanilide(AAPF)SigmaL11211211RT2PCRGIBCOTRIzolJurkatRNA,PCRL11212pBV220LRNA,RNA,A=0.01,;,Goldkey[3,4]L11213pBV220[5],16%SDS2PAGELPBS,,16molöL,8molöL,30kD,,16%SDS2PAGEL11214,[6]:1ml,L865Ll(50mmolöLHepes,100mmolöLNaCl,pH8.0),10Ll700nmolöLhFKBP12(hFKBP12),,100Ll4mgömlA2,25Ll3mmolöLAAPF400mmolöLö,,390nm(A390nm),10sL2211hFKBP12[7]hFKBP12cDNA,hFKBP12LRT2PCR,TJurkathFKBP12,340bp(Fig.1)LpGEMT2easy,,hFKBP12LFig.1AgarosegelelectrophoresisofclonedhFKBP12fromRT2PCRA.RT2PCRproductofhFKBP12B.DL2000marker212hFKBP12RT2PCRpBV220,16%SDS2PAGELpBV220,hFKBP(52GC2GAATTCATATGGGAGTGCAGGTGGAA23),hFKBPL,(52CGAATTCATATGGGTGTTCAGGTTGAAACTATCTCTCCA23),RNA(Fig.2)LpBV220,16%SDS2PAGE14kD,,20%(Fig.3),LJM109hFKBP,hFKBP12,,L30kD,hFKBP12L213PPIasehFKBP12,7nmolöLPPIaseL,hFKBP12PPIase,hFKBP12[7](Fig.4),LL,,L3233:FKBP12Fig.2TheRNAsecondarystructureandfreeenergyanalysisbeforeandafterredesignA.The5endRNAstructurebeforeredesign,thefreeenergyis-36.802kJ;B.The5endRNAstructureafterredesign,thefreeenergyis-15.892kJ;C.The3endRNAstructure,thefreeenergyis-15.857kJFig.3SDS2PAGEanalysisoftherecombinanthFKBP12A.Molecularweightmarker;B.UninducedpBV2hFKBP12öJM109;C.InducedpBV2hFKBP12öJM109;D.hFKBP12proteinafterrenaturation;E.PurifiedhFKBP12proteinFig.4A2chymotrypsincoupledchromogenicassayofPPI2aseactivityofpurifiedhFKBP12A.7nmolöLhFKBP12;B.NohFKBP12,L,,mRNALpBV220,,,,hFKBP12pBV220RNA,hFKBP12L,[8]LhFKBP12FK506,FK506L,hFKBP12,,,L,hFKBP12;hFKBP12,LhFKBP12,L(References)1GoldBG.FK506andtheimmunophilinsinnerveregeneration.MolNeurobiol,1997,15(3):2853062SteinerJP,HamiltonGS,RossDT,ValentineHL,GuoH,ConnollyMA,LiangS,RamseyC,LiJH,HuangW,HoworthP,SoniR,FullerM,SauerH,NowotnikAC,SuzdakP.Neu2rotrophicimmunophilinligandsstimulatestructuralandfunc242316tionalrecoveryinneurodegenerativeanimalmodels.ProcNatlAcadSciUSA,1997,94:201920243,1RNA1(LiWu2ju,WuJia2jin.PredictionofRNAsec2ondarystructurebasedonrandomstackingofhelicalregions.ChinJBiophys),1996,12(2):2132184,1RNA1(LiWu2ju,WuJia2jin.ConstructionofpredictionsystemofRNAsecondarystructure.ProgBiochemBiophys),1996,23(5):4494535,,1PRPL1(ZhangZhi2qing,YaoLi2hong,HouYun2de.Constructionandapplicationofaprocaryotichigh2ex2pressionvectorcontainingPRPLpromoter.ChinJVirol),1990,6(2):1111156KofronJL,KuzmicP,KishoreV,Coln2BonillaE,RichDH.Determinationofkineticconstantsforpeptidyl2prolylcis2transisomerasesbyanimprovedspectrophotometricassay.Biochem2istry,1991,30:612761347StandaertRF,GalatA,VerdineGL,SchreiberSL.MolecularcloningandoverexpressionofthehumanFK5062bindingproteinFKBP.Nature,1990,346(16):6716748,,,1Ricin2AE.coli1(PeiWu2hong,ShenBei2fen,LiWu2ju,HuMei2ru.Computer2aideddesigninhigh2expressionofrecombinantricin2AchaininE.coli.JCellMolImmunol),1998,14(1):333620002000200081820L:11(1);(2);(3);(4);(5);(6)(MHC);(7)L21()L314000,A4,300500L:220():200433;:[e-mail]:200073141900L::::651000503265000113751:1000802714::[phone],:[phone]:[e-mail]@npc.haplink.com.cn5233:FKBP12