Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24026
標題: split-intein 於蛋白質接合作用的應用之探討
Study of the application of split-intein on protein splicing
作者: 洪士勛
Hong, Shi-Xun
關鍵字: split-intein
split-intein
protein splicing
蛋白質接合作用
出版社: 生物化學研究所
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摘要: 藉由Iwai 等人於2006 年發表的期刊中得知,Npu DnaE split-intein應用於免疫球蛋白結合蛋白B1 domain(簡稱為GB1)進行反式蛋白質接合作用可以達到大於98%以上的效率,藉由蛋白質序列比對發現同樣為藍綠菌屬的Npu DnaE split-intein 與Ssp DnaE split- intein 具有高度的相似性,因而以Npu DnaE split-intein 的N 端intein 片段與Ssp DnaE split-intein的C 端intein 片段這樣的組合進行蛋白質反式接合作用效率更佳。 本論文的研究方向主要是以Npu DnaE split-intein 的N 端intein 片段與Ssp DnaE split-intein 的C 端intein 片段之組合,並且進行點突變以探討split-intein 應用於重組具有功能性之大分子量蛋白質的可能性;並加以應用在本實驗室感興趣之三個蛋白上,分別是人類RHA 蛋白,RIG-1 CARDdomain,流感病毒NS1 蛋白。於人類RHA 蛋白之應用是著重於解決RHA 蛋白表現之問題,RIG-1 CARD domain 及流感病毒NS1 蛋白之應用是著重於將原本兩個獨立的domain 藉由intein 的蛋白質接合作用接合而獲得有活性的重組蛋白,此外我將原本分離為N 端及C 端片段的Npu DnaE split-intein 與Ssp DnaE split-intein 以一段spacer 連接構築成融合蛋白也得到極佳的效 率,因而可擴增intein 的應用。
According to the result published in 2006 by Iwai etc, Npu DnaE split-intein have >98% trans-splicing efficiency using non-native extein (GB1 protein), this paper also found that the naturally occurred cyanobacteria split-intein, Npu DnaE split-intein and Ssp DnaE split-intein have highly homology by protein sequence alignment. It is demonstrated that combining the N-terminal Npu DnaE split-intein with the C-terminal Ssp DnaE split-intein can result in a even higher trans-splicingefficiency. In this study, I use point mutation method to modified split-intein junction to facilitate the application of this approach to some protein expression problem . Particularly this modified split-intein module could be used to express large protein that is difficult for expression in E.coli cell. I also apply this module to three proteins under study in our laboratory, including RHA, RIG-1 CARD domains and influenza A virus NS1 protein. Finally, I also found joining the separate split-intein into a fused intein by a linker can further increase protein splicing efficiency, and should be useful for the application of protein splicing in protein expression problems.
URI: http://hdl.handle.net/11455/24026
其他識別: U0005-3107200915591100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3107200915591100
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