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依博素生物合成基因STE15的克隆表达及酶学性质研究摘要A1A0A1A0A2A3A4A5A6A7A8A9A10A11A12STE15A13A14A15A16A17A18A19A20A21STE15A22A23A24A25A26A17A27PET30AA28A29A30A31A32A33BL21DE3A34A35A36A37A38A28A39A40A41A42A43A44A45A45A46A47A48A49A50A13STE15A51A52A35A36A14A53A16A54A55A18A56A57SDSPAGEA58A59A60A61A29A30A6250KDA63A64A65A66A67A68A69A13A51A52A68A70A28A71A72A54A73A74A75A18STE15A51A52A29ECOLIA34A76A77A78A79A80A81A82A83A84A85A86A37A38A28A87A88A89A9050A91A28A92A77A78A85A86A13A51A52A39A93A94A79A95A96A97A49A50A28A98A99A100A67A68A70A51A52A18STE15A51A52A101A102A50UDPA103A104A105A106A107A99A105A22A108A109A82A110A28A111A112A113A114A115A28A42A43A116A117A114A118A13A119A50A120A121A28A122A48A123A124A125A84A53A50A105A22A126A82A127A128A102A50A53A16A18A56A129STEL5A130A131A132A133A134A135A135A136A137A138A139A140A141A142A143A144A145A146A147A148A149A150A151A152A153A154A133A134A135A155A156A135A157A158A159A160A161A162A163A164A137A138A165A166A167A168A167A168A169A170A171A172A173A174A175A176A177A173A178A179A180A181A182A183A184A185A186A187A187A174A188A189A190A191A192A193A194A195A196A197A198A199A200139STREPTOMYCESSP139A201A202A203A204A205A206A207A208A209A210A211A212A213A214A215A216A203IA210A217A218A219A220A221A222A223A224A2251A226A191A192A193A197A227A228A229A230A231A232A229A230A233A234A229A230A235A236A229A230A237A238A239A229A230A240A229A230A241A242A229A243A231A232A229A229A244A245A2468A196A247A229A248A2492A226A191A192A193A202A250A251A249A252A253A254STEA255A167A169A0A219A112A1STE15A252A253A203A10A2A31,269BPA219A172A4A195A5422AAA203A12A63A226A175A7A213A8A9A11A13A112A172A4A201A250A203CA180A181217369AAA182A183A14A15A16PFAM00534A17A18A19A20A23A187A21A22A24A25A26A27A28A291A23A190A28A29A17A22A24A25A26A27A93A30UDPA150ADPA150GDPA191CMPA35A31A17A32A30A22A24A25A26A33A34A36A37A38A23A71A39A22A40A236A196A41A42A22A23A197A43A22A44A198A45A46A47A24A48A49A50A51A52A53A54A55A56A57A24A48A58A59A60A61STREPTOMYCESSP139STE15A2104A211A23A62A63A64A65A66A67A23A190A60A61A133A68A17A43A22A69A68A38EPS15MA23A94A70A22A52A72A73A59A18139A820A82A182A23A214A74A216A75A61STE15CA133A68A17A43A22A69A68A38EPS15CA23A94A70A22A52A72A57A76A77A78A79A80A1810A82108A82A1824A23A131A219A81A83STE15A24A48A84A85A86A87A94A70A22A22A24A25A26A27A23A220A62A63A64A65A17A68A38A88A89A44A198A90A91STE15A24A48A92A95A96A97A98A99A100A101A23A36A35A102A103A104A72A105A105A106A107A83A108A72A109A110A83A78A96A190A111A113A17A27A114A32A115A23A116A78A117A171A94A70A22A22A24A25A26A27A23A119A118A63A64A65A68A38A88A89A120A121A122A123A41A94A70A22A76A124A125A126A127A128A129A130A132A134A135A136A137A1381材料和方法11STE15基因的克隆和表达A139SSP139A224DNAA140A141A142A143A1445GTTCCATGGTCATGCACGTCATGGTGG3NCOIA1455GGCAAGCTTTCATCTGTTTCCCCCATCC3HINDIIIA140A146A147A143A148A149PCRA151A152A143A151A152A153A154A140A22995A2303MINA23194A23045SA14350A23050SA14372A2301MINA143A16430A174A155A156A23172A23010MINA157A234A158A159A160A161A162PET30AA163A165A166A168A143A170A173A176ECOLIBL21DE3A177A178A179A143A205A184A170A173A185A157A170A173A185A186A188A163A165A189A148A149A192A240A193A194A143A195A199A200A160A161A159A201A202A163A165A203A204A140PET30ASTE15A143A201A202A179A206A203A204A140BL21/PET30ASTE15A143A246A148A149A207A208A209A194A157A212BL21/PET30ASTE15A2491A213A215A217A218A195A22125G/MLA250A222A223A225A159LBA252A226A227A14337A230A252A226A176DD600A22804A254A143A232A233IPTGA235A236A237A14005MMOL/LA143A238A2394HA189A143A241A242A179A178A15712重组STE15蛋白的纯化A243A244A245A247A248A251A253A255A32A05ML/GA9A1A135A2A11A2531BINDINGBUFFER50MMOL/LNAH2PO4,300MMOL/LNAC1A2310MMOL/LIMIDAZOLEA23PH80A29A23A12A3A4A30A5A15A6A7A8A1010,000RPMA19A13A14A16A101A21A17A1045MA18A20A201A18A239A22NI2NTAHISBINDRESINS4A230A49A24A201A25A26A27A28A31A101A21A17A101A84A85A33A86A10A34A35A36A3715ML1WASHINGBUFFERA18160MMOL/LA38A39A1005MOL/LNACLA1020MMOL/LTRISHCLA182A40A41A42A28A31A105MLELUTINGBUFFERA18105MOL/LA38A39A1005MOL/LNACLA1020MMOL/LTRISHCLA182A40A43A44A45A28A31A10A46A47A1718MIN/MLA26A48A50A14A16A40A43A17A10A30A5SDSPAGEA48A51A2613STE15阻断株细胞膜的提取A130A52A53A54A55A56A57A58A59A60A249A61KIESERA62A63A64A65A66A675A68A691LSTE15A70A71A72A73A74A75STREPTOMYCESSP139STE15A76A77A63A78A79A80A77A81A82A83A87A223PTCBUFFERA88SUCROSE103,CACL22H2O0386,MGCL26H2O0204,K2SO40025,TESBUFFER025MOL/L,PH728V/V,TRACEELEMENTSOLUTION02V/VA89A90A913A92A93A94A95A96A97A68A94A95A96A97A983300GA99A10010MINA89A102A103A104A66A67A105A106A99A100103000G30MINA102A107A108A89A10968SUCROSEW/WA11020MMPOTASSIUMPHOSPHATEPH78A111A97A89A112A113A77A11425MLA89A115A103A116A117A1182ML42SUCROSEW/WA89197000GA99A10090MINA119A120A99A100A98A63A121A122A123A124A89A10920MMPOTASSIUMPHOSPHATEPH78A111A97A112A125A126A113A127A128A12910A89125000GA99A10030MINA89A119A120A107A108A131A132A94A95A133A89A109100L025MSUCROSE,5MMPOTASSIUMPHOSPHATEPH78A111A97A6814酶活性测定A134A136A137A138A139A140A141A142A143A144A145PALCICA146A147SYLVIEA146A148A149A150A151A1526,7A153A154A155A156A157A22975UPYRUVATEKINASEPKA15815ULACTATEDEHYDROGENASELDHA15807MMOL/LPHOSPHOENOLPYRUVATEA15806MMOL/LNADHA15850MMOL/LKCLA15813MMOL/LHEPESPH70A15813MMOL/LMNCL2,013W/VBSAA158100MOL/LUDPGLUCOSEA158A159A160A161A238A162A163A16415GA165A166A167STE15A168A169A158A170A172A173A154A174A175300LA15337A176A177A17810MINA179A180A183340NMA184A185A186A187ODA167A188A166A1532结果21STEL5基因的克隆和表达A189A190A191A192PET30ASTE15A193A194A241A195A196A197A198FIG1AA199A200A202A203A204A205A206A207A208A209A210A211A212A213A214A215A216A217A218A219A220ECOLIBL21/PET30ASTE15A221A222A199A224A225SDSPAGEA226A227A199A196A197A218A204A228A231A232A233A234A235A236A237A240A242A243A244A245A234A199A246A247A248A25050A251A252A2531BA254A210A255A171A70A203A44A0A232A233A220A1A2A199A71A44A0A235A236A243A220A228A231A232A233A199A34A3A4A5A220A72A6A210FIG1ARESTRICTIONENZYMEANALYSISOFPLASMIDPET30ASTE151MARKERDNA/ECORI2PLASMIDPET30A3PLASMIDPET30ASTE154PET30A/NCOIHINDIII5PET30ASTE15/NCOIHINDIIIFIG1BSDSPAGEANALYSISOFTHELYSATEOFECOLIBL21/PET30ASTE151THELYSATEOFECOLIBL21/PET30AWITHOUTINDUCTION2THELYSATEOFECOLIBL21/PET30ASTE15INDUCEDWITH05MMIPTG3THEMOLECULARWEIGHTMARKER66KD,43KD,31KD4THESUPERNATANTOFTHELYSATEINDUCEDWITH05MMIPTG5THEPELLETSOFTHELYSATEINDUCEDWITH05MMIPTG22重组STEL5蛋白的纯化A45A7HISBINDKITA249A39A8A9A10A42A23A43STE17A125A11A12A13A14A15A16A17A23SDSPAGEA76A18A51A19A20A21ABA5350KDA22A16A17A12A13A181A692A182A24FIG2SDSPAGEANALYSISOFPURIFIEDRECOMBINANTSTE151ECOLIBL21/PET30AWITHOUTINDUCTION2THESUPERNATANTOFECOLIBL21/PET30ASTE15LYSATEINDUCEDWITH05MMIPTG3PURIFIEDRECOMBINANTSTE154MOLECULARWEIGHTMARKER66KD,43KD,31KD23STEL5基因编码蛋白STE15的酶活性测定A25A78A67UDPA94A26A27A28GDPA151A29A27A28TDPLA155A30A27A285A75A31A32A33A35A27A36A37A27A38A40A41A28A67STE15A206A46A47A48STREPTOMYCESSP139STE15A49A50A52A54A192A55A56A57A58A59A60A61A79A62A63A64A65A66A68A73A73A74A222A77A80STE15A81A82A49A83A84A85A86A87A88A89A90A91A92A93UDPA95A96A56A97A93A98A99A100A101A102340NMA103ODA104A49A105A106A61A111A107A108A109A110A112A84A113A56A57A114A60A115A92A47A113A61A89A90STE15A81A82A55A95A96A56A56A57A116A117A83A86A93STE15A81A82A118A74A119A120A12105GA122A123A61340NMA103ODA104A49A105A106A121A77A8010MINA122A124A81A82A118A74A126A177A127A61A128A129A81A82A118A74A130A131ODA104A49A105A106A132A130A131A121FIG3AA122A86A93A56A57A59A60A49A118A74A133A134A98A12114LA122A61A73A135A136A104A49A47A113A124A108A126A137A177A127A141A138A61A139A128A129A56A57A59A60A118A74A140A142A143A130A131510LA61340NMA103A73A135A136A104A144A145A47A113A61A83A146A147A148A149A150A121FIG3BA122A86FIG3AEFFECTOFPROTEINCONCENTRATIONSONTHEREACTIONOFGLYCOSYLTRANSFERASEREACTIONCATALYZEDBYTHEPURIFIEDRECOMBINANTPROTEINSTE15FIG3BEFFECTOFACCEPTORCONCENTRATIONONTHEGLYCOSYLTRANSFERASEREACTIONCATALYZEDBYTHEPURIFIEDRECOMBINANTPROTEINSTE153讨论A175A152A153A154A156A157A158A159A160A161A162A163A164A165A166A167A168A169A170A172A168A173A174A176A178A170A179A179A172A180A163A164A165A183A184A185A186A187A188A189A190A191A193A194A185A195A196A197AB0005010150202503035012345SET15PROTEINGOD/10MINAT340NM0005010150202503035012345678910ACCEPTORLOD/10MINAT340NMA198A199A200A201STE15A202A203A204A205A207A208A209STEL5A210A211A212A213A214A207A215A202A216A217A218A219A220A221A223A207A212A214A224A225A226A227A228A229A230A231A232A233A234A215A235A236A215A202A237A238A239A228A240A202A203A241A242A243A244A245A246A247A248A250A251A247A252A253A254A255A250A1A111A0A2A3A4A5A101A1A247A250A11A6A7A114A29A8A9A207A10A24A235A236A215A202A192A12A238A239A34A35A13A14A15A16A17A17A18A19A20A21A22A23A25A26A27A28A30A31A202A203STE15A204A205A208A209A207A27A28A232A224A225A123A20A21STE15A208A209A46A32A233UDPA94A33A215A216A217A96A215A202A192A12A238A36A239A27A31A102A37A38A239A14A15A105A39A37A40A207A41A233A42A43A239A44A45A47A48A49A50A232A233A215A202A237A238A51A220A32A233A232A224A239A64A179STE15A208A209A52A53A50A94A33A215A215A202A216A217A218A239A119A54A23A25A26A27A28A30A31A29A229A230A231A94A33A215A96A124A55A56A57A58A59A60A61A62A63A65A66A67A68A90A69A125A70A71A243A63A127A72A73A74A75A129A76A77STE15A78A79A130A80A209A81A82A83A55A84A85A86A87A76A88A89A132A57A209A132A57A91A9242A93A67A198A133A95A97A134A98A99A100A103A104A106A135A55A56A57A58A59A63A136A107A108A103A104A106A63A109A110A112A113A138A115A116A117A99A78A141A231A118A117A142A120A121A103A104A106A63A109A110A112A113A76A205A122A126A63A103A104A106A128A109A110A131A75A117A63A145A137A95A139A67A198A127A72A140A139A143A144A146A147A85A148A148A149A150A151A117A99A55A78A65A66A152A63A153A154A76A89A68A75A155A150A81A76A55A78A65A66A152A156A157A158A109A63UDPA69A159A160PYRUVATEKINASEPKA108LACTATEDEHYDROGENASELDHA158A109A63NADHA63A161A157A162A163A1476,7A76A164A165A166A229GLYCOSYLTRANSFERASEUDPGLUCOSEACCEPTORA58A55ACCEPTORUDPPKUDPPHOSPHOENOLPYRUVATEPYRUVATETTPLDHPYRUVATENADHLACTATENADNADHA89340NMA167A131A148A168A169A76A109A158A63NADA170A171A131A76A172A173A159A160A151A117340NMA167A148A168A169A63A165A174A175A176A151A117A167A55A78A65A66A152A63A153A154A67A177A71A55A78A65A66A152A153A154A151A117A155A150A178A131A180A181A182A183A184A185A186A187A188A189A190A191A182A183A184A185A186A193A188A194A195A196A197A199A200A201A202A203A204A180A181A182A183A184A206A207A208A210A211A185A197A193A212A213A214A215A216A206A217A218A197A219A220A195A196A206A221A222A223A193A212A224A225A206A226A217A227A228A230A232A222A195A1
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