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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 | 引用: | Abdel-Ghafar, A. N., Chotpitayasunondh, T., Gao, Z., Hayden, F. G., Nguyen,D. H., de Jong, M. D., et al. (2008). Update on avian influenza a (h5n1)virus infection in humans. N Engl J Med, 358(3), 261-273. Aslanidis, C., & de Jong, P. J. (1990). Ligation-independent cloning of pcr products (lic-pcr). Nucleic Acids Res, 18(20), 6069-6074. Beigel, J. H., Farrar, J., Han, A. M., Hayden, F. G., Hyer, R., de Jong, M. D., et al. (2005). Avian influenza a (h5n1) infection in humans. N Engl J Med,353(13), 1374-1385. Bornholdt, Z. A., & Prasad, B. V. (2008). X-ray structure of ns1 from a highly pathogenic h5n1 influenza virus. Nature, 456(7224), 985-988. Buskirk, A. R., Ong, Y. C., Gartner, Z. J., & Liu, D. R. (2004). Directed evolution of ligand dependence: Small-molecule-activated protein splicing. Proc Natl Acad Sci U S A, 101(29), 10505-10510. Demidov, V. V., & Broude, N. E. (2006). 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(2003). A genetic approach to identifying mitochondrial proteins. Nat Biotechnol, 21(3),287-293. Paulus, H. (1998). The chemical basis of protein splicing. Perler, F. B. (2006). Protein splicing mechanisms and applications. IUBMB Life,58(1), 63. Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., et al. (1994). Protein splicing elements: Inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res, 22(7), 1125-1127. Perler, F. B., Olsen, G. J., & Adam, E. (1997). Compilation and analysis of intein sequences. Nucleic Acids Res, 25(6), 1087-1093. Pietrokovski, S. (1994). Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins.Protein Sci, 3(12), 2340-2350. Romanelli, A., Shekhtman, A., Cowburn, D., & Muir, T. W. (2004).Semisynthesis of a segmental isotopically labeled protein splicing precursor: Nmr evidence for an unusual peptide bond at the n-extein-intein junction. 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FEBS Lett, 583(5), 909-914. | 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 |
| Appears in Collections: | 生物化學研究所 |
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