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標題: 經由雙精胺酸轉位路徑輸送基因重組蛋白質之研究
Translocation of recombinant protein via the Tat pathway in Escherichia coli
作者: 葉志賢
Ye, Jhih-Sian
關鍵字: Twin-arginine translocation;雙精胺酸轉位系統;signal peptide;Green fluorescence protein;Secretion;TorD;訊息導引胜肽綠色螢光蛋白
出版社: 化學工程學系所
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大腸桿菌有多種路徑輸送其分泌蛋白,近年來發現新興系統,雙精胺酸轉位(twin-arginine translocation)系統(Tat),能夠輸送已摺疊好之蛋白至細胞間質。然而,Tat系統之輸送效益比起Sec系統還要來得低。在先前地研究發現到共表現TorD能夠增進有連接TorA之訊息導引胜肽的綠色螢光蛋白(GFP)。此研究之目的找出GFP蛋白和TorD蛋白之間最佳化之產出比例。在最後,製作好三種基因重組蛋白之培養基含有可產TorD蛋白和GFP蛋白。細胞質與細胞間質可藉由細胞劃分來做分離,西方墨點法與螢光光度計來做為蛋白輸送效益之依據。

Gram-negative bacteria such as Escherichia coli have multiple pathways for exporting secretory proteins. The Twin-arginine translocation (Tat) pathway was recently found to be a novel system which is capable of translocating folded proteins into the periplasm. However, the translocation efficiency of the Tat pathway is not as high as that of the Sec pathway. It has been previously shown that the co-expression of TorD is capable of enhancing the translocation of green fluorescence protein (GFP) with TorA signal peptide. One of the objectives of this study is to identify the optimal stoichiometric ratio between GFP and TorD for protein secretion. To this end, three recombinant E. coli strains harboring plasmids encoding GFP fusion and TorD were constructed. Periplasmic and cytoplasmic fractions of the cells were fractionated by sucrose gradient centrifugation. Western blotting analysis and fluorescence spectroscopy were used to evaluate the efficiency of protein translocation.
其他識別: U0005-2708200710442900
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