49969.β1，4半乳糖基轉移酶在畢赤酵母中的克隆、表達及活性測定

1、洳專：3碩士學位論文⑧論文題目笪：!，壘：坐堊l趨基整整醒壟望麥酵母中的克隆、表達及活性測定作者姓名指導教師學科(專業(yè))所在學院全蒸龔送國熬援—生物絲堂：皇坌王生塑堂生金抖堂堂瞳⑧一Abstract伊l，4一Galactosyltransferase—I，伊1，4一GTI，catalyzesthetransferofgalactose(Gal)fromUDP—GaltotheNacetylglucosamine(GlcNAc)resid

2、uepresentatthenonreducingterminalendofglycansofglycoproteinsandglycolipids，producingp一1，4linkedgalactosylatedglycanInadditiontoGlcNAcasanacceptortheenzymecanalsouseothersugars，suchasNacylsubstitutedglucosaminesandNacetyl

3、Dmannosamine，asacceptorsPichiapastorishererologonsgeneexpressionsystemhasbeenutilizedtoproduceattractivelevelsofavarietyofintracellularandextracellularproteinsItwillbeemployedmorewidelyforitsmanyadvantages，includingfastc

4、ulture，cheapmedium，easyoperating，activeproductsbysecretedexpression，proteinprocessing，proteinfolding，posttranslationalmodification，easyindustrialproductionandSOonInthispapertheeukaryotieexpressionofthe014GTgenebyPichiapa

5、storishererologousgeneexpressionsystemWasstudiedThe8—1，4一GTgenewasgotfromtheplasmidpGEX4T_2throughDTPCRtechniques口1，4一GTgenewasinsertedintoaPichiapastorisvectorpPIC9K(witha—factorsignal)constructingarecombinantexpression

6、plasmidpPIC9K—GTbygettingatandemofmultiplecopiesTherecombinantplasmidwastransformedintoPichiapastoriscelllineGSll5byelectroporationThetransformantswerescreened，culturedandinducedbytheadditionofO5％(v／v、methan01TheSDS—PAGE

7、analysisshowedthattheweightofthefusionproteinwascorrectTheexpressionof伊1，4GTreachedupto35％ofthetotalproteinsinmediumsupematantasshownbySDS—PAGE伊1，4GTwaspurifiedbygelfiltrationusingSephadexG一75columnWeappliedthepH—indicat

8、orbasedassaytodetectactivityTheactiveunitis986，specificactivityis2143U／mg盧1，4一GTwithbiologicalactivitywasexpressed，purifiedandindentifiedforthefutherstudyorpharmacologyapplicationKeywords：∥l，4一galactosyltransferase，Eukar