Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3695
標題: 共表現伴護蛋白對大腸桿菌雙精胺酸轉位系統之影響
Enhanced Translocation of Recombinant Proteins via the Tat Pathway with Chaperones in Escherichia coli
作者: 李亞芳
Lee, Ya-Fang
關鍵字: 伴護蛋白
Chaperone
雙精胺酸轉位系統
Tat pathway
出版社: 化學工程學系所
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摘要: 雙精胺酸轉位系統(Twin-arginine translocation)為一新穎之蛋白輸送系統,其特色為可選擇性將摺疊完成之蛋白自細胞質區輸送至細胞間質區。因此利用此系統表現基因重組蛋白為一具有前瞻性之策略。然而,過去的文獻指出,利用大腸桿菌雙精胺酸轉位系統輸送基因重組蛋白質的效率往往不甚理想,以致於雙精胺酸轉位系統未能被廣泛利用。本研究利用共表現DmsD蛋白增進利用雙精胺酸轉位系統輸送N端接有DmsA訊息引導胜肽的綠色螢光融合蛋白之效率。根據螢光強度分析顯示,相較於未共表現DmsD蛋白之處理,共表現DmsD蛋白可增進綠色螢光融合蛋白之轉位達28.56%。之所以可增加雙精胺酸轉位系統之效率則推測原因為DmsD蛋白可減緩綠色螢光融合蛋白被蛋白水解酶水解。透過共表現DmsD蛋白對於N端接有TorA訊息引導的綠色螢光融合蛋白系統以及共表現TorD對於N端接有DmsA訊息引導胜肽的綠色螢光融合蛋白系統之螢光強度實驗顯示其分別達15.01%及31.79%的提升效果,表示共表現DmsD蛋白及TorD蛋白皆可增進雙精胺酸轉位系統的輸送效率及DmsD蛋白、TorD蛋白與其專一性雙精胺酸訊息導引胜肽間存在交叉作用關係。其中又以共表現TorD蛋白增進效果最好,此與其更能有效降低蛋白被水解關係密切。
Twin-arginine translocation (Tat) pathway is capable of translocating folded proteins into the periplasm of Gram-negative bacteria and thus holds great potentials for the expression of recombinant proteins in Escherichia coli Nevertheless, this promise has been hampered by the characteristic low translocation efficiency. In this study, it is detected that the coexpression of DmsD, a cytoplasmic chaperone similar to TorD, in conjunction with the DmsA signal peptide can enhance the translocation of GFP fusion by 28.56% probably by reducing proteolysis via its binding to the signal peptide. Besides, coexpression of DmsD in conjunction with the TorA signal peptide and coexpression of TorD with the DmsA signal peptide can also get the enhancement of 15.01% and 31.79% respectively meaning the presence of cross-reactivity between DmsD and TorD and the stronger cross-reactivity exhibited by TorD. The reason for the stronger cross-reactivity might be attributed to the strong chaperone activity of TorD against proteolysis.
URI: http://hdl.handle.net/11455/3695
其他識別: U0005-2104200813050500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2104200813050500
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