Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52221
標題: 小球藻穀氧還原蛋白結構與生化特性分析
Structural and functional relation analysis of glutaredoxin from Chlorella sorokiniana T89
作者: 陳亭妏
Chen, Ting-Wen
關鍵字: 小球藻
Chlorella
穀氧還原蛋白
結構與生化特性
glutaredoxin
structural and functional relation
出版社: 食品暨應用生物科技學系所
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摘要: 小球藻Chlorella sorokiniana T89(CsT89)之抗氧化酵素—穀氧還原蛋白(Glutaredoxin, Grx)已被成功選殖並且完成生化特性分析。為了進一步瞭解其結構與生化特性間之相關性,本實驗將小球藻穀氧還原蛋白之榖胱甘肽(glutathione, GSH)結合位以及保留性序列之胺基酸位置進行點突變,並針對其結構與催化活性之關係作探討。 實驗設計七個突變位置,共十二個點突變重組蛋白rCsT89 GrxK22R、rCsT89 GrxIQ5859TK、 rCsT89 GrxQ59K、rCsT89 GrxQ59N、rCsT89 Grx68PV、rCsT89 GrxS70T、rCsT89 GrxG84A、rCsT89 GrxG84N、rCsT89 GrxG84T、rCsT89 GrxG84Y、rCsT89 GrxD85E與rCsT89 GrxD85T,以蛋白質表現宿主E. coli BL21(DE3)誘導表現,rCsT89 GrxK22R、rCsT89 GrxQ59K、rCsT89 GrxS70T、rCsT89 GrxG84A、rCsT89 GrxG84Y、rCsT89 GrxD85E與rCsT89 GrxD85T等七個重組蛋白,可大量表現可溶性蛋白質,經由金屬離子親和性管柱純化上述目標重組蛋白,並利用HED (β-hydroxyethyl disulfide) assay進行生化特性分析,結果顯示各個點突變重組蛋白其最適反應pH值為pH 8.5-9;最適反應溫度介於40-55°C;rCsT89 GrxG84A以及rCsT89 GrxD85E之酵素活性略高於rCsT89 Grx之活性,而rCsT89 GrxK22R、rCsT89 GrxQ59K、rCsT89 GrxG84Y以及rCsT89 GrxD85T活性則降至CsT89 Grx之一半,rCsT89 GrxQ59N與rCsT89 GrxS70T活性僅剩約20%。進一步分析點突變重組蛋白之酵素動力學rCsT89 GrxQ59K、rCsT89 GrxG84A與rCsT89 GrxD85E之kcat與Km值皆上升,然而rCsT89 GrxD85T則是kcat值下降且Km值上升。實驗亦發現rCsT89 Grx突變成rCsT89 GrxK22R與rCsT89 GrxG84Y時會使酵素穩定性下降,影響實驗之再現性。
The gene of glutaredoxin (Grx) from Chlorella sorokiniana T89 (CsT89) has been cloned and overexpressed successfully in E. coli. The recombinant protein was biochemically characterized. In order to realize the structural and functional relation, in this study, based on the glutathione (GSH) binding sites and conserved residues of glutaredoxin, 12 single point mutations were designed from CsT89 Grx. Twelve mutations were designed as the following rCsT89 GrxK22R, rCsT89 GrxIQ5859TK, rCsT89 GrxQ59K, rCsT89 GrxQ59N, rCsT89 Grx68PV, rCsT89 GrxS70T, rCsT89 GrxG84A, rCsT89 GrxG84N, rCsT89 GrxG84T, rCsT89 GrxG84Y, rCsT89 GrxD85E and rCsT89 GrxD85T. The protein expressions of recombinant mutant glutaredoxins were induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli BL21(DE3). Target recombinant soluble proteins were purified by TALONR metal affinity resin and analyzed by 16% tricine SDS-PAGE. The activities of recombinant mutant glutaredoxins were determined by the HED (β-hydroxyethyl disulfide) assay. The optimal reaction pH and temperature of recombinant mutant glutaredoxins were pH 8.5-9 and 40-55°C, respectively. Compare with rCsT89 Grx activity, CsT89 GrxG84A and rCsT89 GrxD85E activities were slightly higher; rCsT89 GrxK22R, rCsT89 GrxQ59K, rCsT89 GrxG84Yand rCsT89 GrxD85T activities were less than 50% of CsT89 Grx activity; activities of rCsT89 GrxQ59N and rCsT89 GrxS70T were only remaining 20% of rCsT89 Grx. Analyze the kinetic studies of recombinant mutant glutaredoxins, both kcat and Km of rCsT89 GrxQ59K, rCsT89 GrxG84A and rCsT89 GrxD85E have elevated. However, kcat of rCsT89 GrxD85T has declined and Km has increased. Furthermore, rCsT89 Grx was mutated to rCsT89 GrxK22R and rCsT89 GrxG84Y caused the low stability of recombinant proteins and effected the reproducibility of experiments.
URI: http://hdl.handle.net/11455/52221
其他識別: U0005-2207201313033100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2207201313033100
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