基于定点突变技术的家蚕酪氨酸羟化酶结构与功能的分析-structure and function analysis of silkworm tyrosine hydroxylase based on site-directed mutagenesis technology.docx

Studiesonthestructureandfunctionoftyrosinehydroxylase withsite-directedmutagenesisinBombyxmoriStudies onthestructureandfunction oftyrosine hydroxylasewithsite-directedmutagenesisinsilkworm, BombyxmoriABSTRACTTyrosinehydroxylaseisanaromaticaminoacidehydroxylase,whichcould catalysistheformationofL-doba,usingL-tyrosineasitssubstrateandcombiningwith tetrahydrobiopterinandFe2+.Therate-limitingstepin thebiosynthesis ofthe catecholaminesanditsderivatives.L-dopawastheprecursorsofsomebiologically activecompounds,includingdopamine,melanin,adrenalineandothersubstances.Then Dopaiscatalyzedtocatecholaminesanditsderivativeswhichparticipateincuticletanningandmelanization.Wt-THexistsinhuman、mammalsandinsetsbeingsasanessentialenzymeformetabolism.Itis thereforehighlysignificanttoresearch structure、functionandstabilityoftyrosinehydroxylase.HerethisstudytakesgonadofBombyx moriformaterials.FirstweanalysisthemajoropenreadingframessequenceofBmTH (geneofTH),thenbioinformatics analysissequenceofBmTH.Afterwards we construct theexpressionvectorandputthemintoE.coliBL-21(DE3),andletthemexpress recombinantproteins.Andfinally,wegotthetargetproteinwithaffinity chromatography.Onthatbasis,wefurtherhadanalyzedthecharacterization,parametersofenzymekinetics,bindingcapacityofsubstrate,function、structureandstabilityoftyrosinehydroxylaseandmutationsfrombombyxmori.Themaincontentandthe results asfollowing:Thecloning、expressionandassessmentofBmTHTheBmTHwasclonedintotheexpressionvectorpET28a(+)byRT-PCRand PCR,andinducedtheconcentrationexpressionandtemperatureexpressionbyIPTGin EscherichiacoliBL21(DE3).Theresultsshowedthattheactivityofproteinexpression couldattainthelargestamountwhentheEscherichiacoliBL21(DE3)inducedby1mMIPTGand16℃.AndtheresultsofSDSandwestern-blotyingindicatedthatwegot63.3KDfusionproteins.Thiswilllaythefoundationforfurtherresearcheson enzymaticcharacterizationandfounctionofBmTHinvitro.Properties ofrecombinantTHStudiesonthestructureandfunctionoftyrosinehydroxylase withsite-directedmutagenesisi