Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24026
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dc.contributor蔡慶修zh_TW
dc.contributorChing-Hsiu Tsaien_US
dc.contributor陳宏文zh_TW
dc.contributorHung-wen Chenen_US
dc.contributor.advisor張功耀zh_TW
dc.contributor.advisorKung-Yao Changen_US
dc.contributor.author洪士勛zh_TW
dc.contributor.authorHong, Shi-Xunen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T07:21:49Z-
dc.date.available2014-06-06T07:21:49Z-
dc.identifierU0005-3107200915591100zh_TW
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Profluorescent protein fragments forfast bimolecular fluorescence complementation in vitro. Nat Protoc, 1(2),714-719. Evans, T. C., Jr., Martin, D., Kolly, R., Panne, D., Sun, L., Ghosh, I., et al.(2000). Protein trans-splicing and cyclization by a naturally split intein from the dnae gene of synechocystis species pcc6803. J Biol Chem,275(13), 9091-9094. Evans, T. C., Jr., & Xu, M. Q. (1999). Intein-mediated protein ligation:Harnessing nature''s escape artists. Biopolymers, 51(5), 333-342. Fukuda, T., Ohta, K., & Ohya, Y. (2006). Investigation of the mechanism of meiotic DNA cleavage by vma1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site. Eukaryot Cell, 5(6), 981-990. Gack, M. U., Kirchhofer, A., Shin, Y. C., Inn, K. S., Liang, C., Cui, S., et al. (2008). Roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction. Proc Natl Acad Sci U S A, 34 105(43), 16743-16748. Ghosh, I., Sun, L., & Xu, M. Q. (2001). Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*. J Biol Chem, 276(26), 24051-24058. Gimble, F. S., & Thorner, J. (1992). Homing of a DNA endonuclease gene by meiotic gene conversion in saccharomyces cerevisiae. Nature, 357(6376), 301-306. Hirata, R., Ohsumk, Y., Nakano, A., Kawasaki, H., Suzuki, K., & Anraku, Y. (1990). Molecular structure of a gene, vma1, encoding the catalytic subunit of h(+)-translocating adenosine triphosphatase from vacuolar membranes of saccharomyces cerevisiae. J Biol Chem, 265(12), 6726-6733. Iwai, H., Zuger, S., Jin, J., & Tam, P. H. (2006). Highly efficient protein trans-splicing by a naturally split dnae intein from nostoc punctiforme. FEBS Lett, 580(7), 1853-1858. Kaluz, S., & Flint, A. P. (1994). Ligation-independent cloning of pcr products with primers containing nonbase residues. Nucleic Acids Res, 22(22),4845. Mills, K. V., Lew, B. M., Jiang, S., & Paulus, H. (1998). Protein splicing in trans by purified n- and c-terminal fragments of the mycobacterium tuberculosis reca intein. Proc Natl Acad Sci U S A, 95(7), 3543-3548. Mootz, H. D., Blum, E. S., Tyszkiewicz, A. B., & Muir, T. W. (2003). Conditional protein splicing: A new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc, 125(35), 10561-10569. Mootz, H. D., & Muir, T. W. (2002). Protein splicing triggered by a small molecule. J Am Chem Soc, 124(31), 9044-9045. Otomo, T., Ito, N., Kyogoku, Y., & Yamazaki, T. (1999). Nmr observation of selected segments in a larger protein: Central-segment isotope labeling through intein-mediated ligation. Biochemistry, 38(49), 16040-16044. Ozawa, T., Nogami, S., Sato, M., Ohya, Y., & Umezawa, Y. (2000). A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal Chem, 72(21), 5151-5157. Ozawa, T., Sako, Y., Sato, M., Kitamura, T., & Umezawa, Y. (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. Proc Natl Acad Sci U S A, 101(17), 6397-6402. Seyedsayamdost, M. R., Yee, C. S., & Stubbe, J. (2007). Site-specific incorporation of fluorotyrosines into the r2 subunit of e. Coli ribonucleotide reductase by expressed protein ligation. Nat Protoc, 2(5),1225-1235. Skretas, G., & Wood, D. W. (2005). Regulation of protein activity with small-molecule-controlled inteins. Protein Sci, 14(2), 523-532. Southworth, M. W., Benner, J., & Perler, F. B. (2000). An alternative protein splicing mechanism for inteins lacking an n-terminal nucleophile. Embo J, 19(18), 5019-5026. Tan, L. P., Lue, R. Y., Chen, G. Y., & Yao, S. Q. (2004). Improving the intein-mediated, site-specific protein biotinylation strategies both in vitro and in vivo. Bioorg Med Chem Lett, 14(24), 6067-6070. Tavassoli, A., & Benkovic, S. J. (2007). Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in e. Coli. Nat Protoc, 2(5), 1126-1133. Telenti, A., Southworth, M., Alcaide, F., Daugelat, S., Jacobs, W. R., Jr., & Perler, F. B. (1997). The mycobacterium xenopi gyra protein splicing element: Characterization of a minimal intein. J Bacteriol, 179(20), 6378-6382. Wu, W. Y., Mee, C., Califano, F., Banki, R., & Wood, D. W. (2006). Recombinant protein purification by self-cleaving aggregation tag. Nat Protoc, 1(5), 2257-2262. Xu, M. Q., & Evans, T. C., Jr. (2001). Intein-mediated ligation and cyclization of expressed proteins. Methods, 24(3), 257-277. Zettler, J., Schutz, V., & Mootz, H. D. (2009). The naturally split npu dnae intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett, 583(5), 909-914.en_US
dc.identifier.urihttp://hdl.handle.net/11455/24026-
dc.description.abstract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。zh_TW
dc.description.abstractAccording 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.en_US
dc.description.tableofcontents目錄................................................................................................................................I 圖表目錄......................................................................................................................III 中文摘要.......................................................................................................................V 英文摘要......................................................................................................................VI 第一章緒論.................................................................................................................1 1.intein與蛋白質接合作用..............................................................................1 1.1 intein的發現與命名..................................................................1 1.2 intein的基本組成..............................................................................1 1.3蛋白質接合 (protein splicing) 作用機制....................................2 1.4 Intein的種類......................................................................................3 1.4.1 maxi-intein..............................................................................3 1.4.2 mini-intein..............................................................................3 1.4.3 丙氨酸 intein..........................................................................3 1.4.4 split-intein............................................................................4 2. Ligation-independent cloning of PCR products(LIC-PCR) .....4 第二章 材料與方法...................................................................................................5 2.1 使用LIC-PCR方式構築質體...................................................................5 2.1.1 引子設計及模板製備........................................................................5 2.1.2 聚合酶連鎖反應(polymerase chain reaction) 及DNA 純化.....5 2.1.3 T4 DNA 聚合酶處理目標基因及載體...............................................6 2.1.4 目標基因與載體接合........................................................................6 2.1.5 質體的轉型........................................................................................6 2.1.6 點突變質體的構築.............................................................................6 2.2 於大腸桿菌細胞內進行蛋白質接合作用分析...................................6 2.3 於小麥胚芽並行式的轉錄與轉譯反應系統中進行蛋白質接合.......7 第三章實驗結果.........................................................................................................8 3.1 蛋白質剪接作用及點突變分析.................................................................8 3.1.1 Npu DnaE split-intein 蛋白質接合作用的效率.......................8 3.1.2 分析不同split-intein 組合的反式接合作用效率......................8 3.1.4 分析點突變對反式接合效率造成的影響......................................8 II 3.2 split-intein的應用................................................................................9 3.2.1 split-intein 突變株應用於大分子量蛋白的表現及重組之探 討 9 3.2.2 RIG1 蛋白的CARD domain 進行蛋白質接合作用之探討...........9 3.2.3 CARD domain 與GB1 蛋白進行交錯蛋白質接合作用之探討...10 3.2.4 流感NS1 蛋白進行蛋白質接合作用之探討..............................11 3.2.5 增進蛋白質接合作用應用性之探討..........................................11 第四章 討論...............................................................................................................13 4.1 split-intein突變株於大分子量蛋白質接合作用之探討............13 4.2 探討RIG-1 CARD domain進行蛋白質接合作用之方法.................13 4.3 以流感NS1蛋白進行蛋白質接合作用的效率之探討.....................15 第五章 參考文獻.......................................................................................................33 第六章 附錄...............................................................................................................36 附錄一. 實驗中所使用的引子........................................................................36 附錄二. 引子設計及模板來源..........................................................................39 附錄三. 各重組質體之構築..............................................................................40 附錄四. 點突變質體構築:................................................................................42 附錄五. 實驗中用到的蛋白質序列及結構:....................................................42 5.1 Human RIG1 sequence (1-284 胺基酸序列): .......................................42 5.2 Human RIG1 蛋白質二級結構預測:....................................................42 5.3 NS1 (H1N1 strain,S103F/I106M/d 80-84)全長胺基酸序列................43 5.4 X-ray Structure of H5N1 NS1: ..............................................................43 附錄六.Intein的應用......................................................................................44 一. EPL 的作用機制及應用:.............................................................44 二. PTS 的作用機制及應用:.............................................................45 圖表目錄 【圖一】蛋白質接合作用..........................................................................................16 【圖二】intein 結構區域(domain)及命名法則.....................................................16 【圖三】蛋白質接合 (protein splicing) 作用機制..........................................17 【圖四】標準的蛋白質剪接作用與替代的蛋白質剪接作用之比較......................18 【圖五】反式蛋白質接合作用..................................................................................19 【圖六】反式蛋白質接合作用機制..........................................................................20 【圖七】LIC-PCR 構築質體的流程圖.......................................................................21 【圖八.a】Npu DnaE split-intein(Iwai et al.,2006)....................................22 【圖八.b】SDS-PAGE 分析Npu DnaE split-intein 反式接合作用.....................22 【圖九.a】Npu DnaE 與Ssp DnaE split-intein 蛋白質序列排比.....................23 【圖九.b】SDS-PAGE 分析不同split-intein 組合的反式接合作用..................23 【圖十】SDS –PAGE 分析點突變對反式接合效率造成的影響.............................24 【圖十一.a】人類RHA 蛋白......................................................................................25 【圖十一.b】構築RHA split-intein 載體............................................................25 【圖十一.c】SDS –PAGE 分析重組RHA 蛋白於小麥胚芽並行式的轉錄與轉譯系 統中表現蛋白的結果。..............................................................................................25 【圖十二.a】RIG1 蛋白的CARD domain..................................................................26 【圖十二.b】構築CARD1 與CARD2 split-intein 載體........................................26 【圖十三】 SDS –PAGE 分析CARD1-IE-IN 與IC-SWD-CARD2 92-284 (A),或 IC-CFN-CARD2 92-284 (B)於小麥胚芽並行式的轉錄與轉譯反應系統中進行蛋白 質接合作用的結果。..................................................................................................27 【圖十四】SDS –PAGE 分析his-CARD1-IE-IN與IC-CFN-GB1 於E.coli BL21 進 行蛋白質接合作用的結果..........................................................................................28 【圖十五】GB1-GS-IN與IC-SWD-CARD2 92-284(A)或IC-CFN-...........................29 【圖十六.a】構築[N-NS1]-GS-[IN]與[IC]-CFN-[C-NS1]融合蛋白....................29 【圖十六.b】SDS –PAGE 分析重組NS1 蛋白於小麥胚芽並行式的轉錄與轉譯系 統中表現蛋白的結果。..............................................................................................29 【圖十七.a】構築GB1-GS-IN-spacer-IC-CFN-GB1 融合蛋白.............................30 【圖十七.b】GB1-GS-IN-spacer-IC-CFN-GB1融合蛋白(A)或GB1-GS-IN-........30 【圖十八】SDS –PAGE 分析GB1-GS-IN-spacer-IC-CFN-GB1 融合蛋白,與 GB1-GS-IN-spacer-IC-AFN-GB1 融合蛋白,於小麥胚芽並行式的轉錄與轉譯反應 系統中進行蛋白質表現的結果。..............................................................................31 【圖十九.a】構築[N-NS1]-GS-[IN-spacer-IC]-CFN-[C-NS1]融合蛋白............31 【圖十九.b】SDS –PAGE 分析[N-NS1]-GS-[IN-spacer-IC]-CFN-[C-NS1].......31 【圖二十】RHA 蛋白二級結構預測結果...................................................................32 【附圖一】IPL / EPL 原理......................................................................................47 【附圖二】蛋白質反式接合作用於蛋白質環化的應用..........................................48 【附圖三】CPS(conditional protein splicing)................................................48 【附圖四】apaf-1,rig-1,mda-5 之CARD domain 多重序列排比之結果...........49 【附圖五】apaf-1 之結構.........................................................................................49zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3107200915591100en_US
dc.subjectsplit-inteinen_US
dc.subjectsplit-inteinzh_TW
dc.subjectprotein splicingen_US
dc.subject蛋白質接合作用zh_TW
dc.titlesplit-intein 於蛋白質接合作用的應用之探討zh_TW
dc.titleStudy of the application of split-intein on protein splicingen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
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