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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張世宗 | |
dc.contributor.author | Li-Hsien Huang | en |
dc.contributor.author | 黃俐嫻 | zh_TW |
dc.date.accessioned | 2021-06-08T05:02:26Z | - |
dc.date.copyright | 2010-08-20 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-19 | |
dc.identifier.citation | Azuma, Y., S. H. Tan, et al. (2001). 'Expression and regulation of the mammalian SUMO-1 E1 enzyme.' FASEB J 15(10): 1825-1827.
Boggio, R. and S. Chiocca (2005). 'Gam1 and the SUMO pathway.' Cell Cycle 4(4): 533-535. Boggio, R., R. Colombo, et al. (2004). 'A mechanism for inhibiting the SUMO pathway.' Mol Cell 16(4): 549-561. Boggio, R., A. Passafaro, et al. (2007). 'Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation.' J Biol Chem 282(21): 15376-15382. Bohren, K. M., V. Nadkarni, et al. (2004). 'A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus.' J Biol Chem 279(26): 27233-27238. Bradford, M. M. (1976). 'A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.' Anal Biochem 72: 248-254. Chiocca, S. (2007). 'Viral control of the SUMO pathway: Gam1, a model system.' Biochem Soc Trans 35(Pt 6): 1419-1421. Deyrieux, A. F. and V. G. Wilson (2009). Viral Interplay with the Host Sumoylation System: 315-329. Dohmen, R. J., R. Stappen, et al. (1995). 'An essential yeast gene encoding a homolog of ubiquitin-activating enzyme.' J Biol Chem 270(30): 18099-18109. Fukuda, I., A. Ito, et al. (2009). 'Ginkgolic acid inhibits protein SUMOylation by blocking formation of the E1-SUMO intermediate.' Chem Biol 16(2): 133-140. Gong, L., B. Li, et al. (1999). 'Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex.' FEBS Lett 448(1): 185-189. Hay, R. T. (2005). 'SUMO: a history of modification.' Mol Cell 18(1): 1-12. Johnson, E. S. (2004). 'Protein modification by SUMO.' Annu Rev Biochem 73: 355-382. Johnson, E. S., I. Schwienhorst, et al. (1997). 'The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.' EMBO J 16(18): 5509-5519. Kerscher, O., R. Felberbaum, et al. (2006). 'Modification of proteins by ubiquitin and ubiquitin-like proteins.' Annu Rev Cell Dev Biol 22: 159-180. Kurepa, J., J. M. Walker, et al. (2003). 'The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress.' J Biol Chem 278(9): 6862-6872. Loftus, L. T., R. Gala, et al. (2009). 'Sumo-2/3-ylation following in vitro modeled ischemia is reduced in delayed ischemic tolerance.' Brain Res 1272: 71-80. Lois, L. M. and C. D. Lima (2005). 'Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1.' EMBO J 24(3): 439-451. Melchior, F. (2000). 'SUMO--nonclassical ubiquitin.' Annu Rev Cell Dev Biol 16: 591-626. Novatchkova, M., R. Budhiraja, et al. (2004). 'SUMO conjugation in plants.' Planta 220(1): 1-8. Saracco, S. A., M. J. Miller, et al. (2007). 'Genetic analysis of SUMOylation in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is essential.' Plant Physiol 145(1): 119-134. Schulman, B. A. and J. W. Harper (2009). 'Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways.' Nat Rev Mol Cell Biol 10(5): 319-331. Tang, Z., C. M. Hecker, et al. (2008). 'Protein interactions in the sumoylation cascade: lessons from X-ray structures.' FEBS J 275(12): 3003-3015. Tatham, M. H., E. Jaffray, et al. (2001). 'Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.' J Biol Chem 276(38): 35368-35374. Uchimura, Y., M. Nakamura, et al. (2004). 'Overproduction of eukaryotic SUMO-1- and SUMO-2-conjugated proteins in Escherichia coli.' Anal Biochem 331(1): 204-206. Uchimura, Y., M. Nakao, et al. (2004). 'Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway.' FEBS Lett 564(1-2): 85-90. Voller, D. and H. Schindelin (2010). 'And yet it moves: active site remodeling in the SUMO E1.' Structure 18(4): 419-421. Walden, H., M. S. Podgorski, et al. (2003). 'The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1.' Mol Cell 12(6): 1427-1437. Wang, Y., I. Ladunga, et al. (2008). 'The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii.' Genetics 179(1): 177-192. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23492 | - |
dc.description.abstract | SUMO (small ubiquitin-like modifier) 是一種類泛素蛋白質。其可以藉由共價結合的方式接到目標蛋白質上,以進行轉譯後修飾作用。SUMO化修飾作用需經過一連串由SUMO E1活化酶、E2銜接酶、E3黏合酶所進行的酵素反應。E1活化酶是由SAE1 (sumo activating enzyme subunit 1) 與SAE2 (sumo activating enzyme subunit 2) 兩個次單元體所組成,並負責SUMO化的起始步驟。其功能為將SUMO活化並轉移至E2銜接酶上。
本論文先以酵母菌、人類及阿拉伯芥的SUMO E1活化酶序列進行BLAST分析,並利用AUGUSTUS軟體預測exon-intron找出可能為單胞綠藻 (Chlamydomonas reinhardtii) SUMO E1活化酶的序列。利用專一性引子,本論文成功選殖出SAE1次單元體及SAE2次單元體之部分序列。嘗試不同的表現與純化條件發現,在有GST (glutathione S-transferase) 協助摺疊的情況下,可得到正確表現之重組蛋白。而無論直接使用CrSAE2部分序列或加上CrSAE1和CrSAE2部分序列一起進行in vitro pull down實驗,皆沒有觀察到其與CrSUMO96-GG間之交互作用。本論文亦將表現情形較佳之GST-SAE2部分序列做為抗原,製備SAE2次單元體之多株抗體,以作為未來研究的工具。 | zh_TW |
dc.description.abstract | SUMO (small ubiquitin-like modifier) is one of the ubiquitin-like proteins, which can covalently attach to target proteins for post-translational modification. SUMOylation cascade includes SUMO E1 activating enzyme, E2 conjugating enzyme and E3 ligases. SUMO E1 activating enzyme is a heterodimer composed of SAE1 (sumo activating enzyme subunit 1) and SAE2 (sumo activating enzyme subunit 2).
I initially performed the BLAST search by using the SUMO E1 sequences of Saccharomyces cerevisiae, Arabidopsis thaliana and human as the template, and predicted the potential exon-intron regions by AUGUSTUS program to find out the potential E1 activating enzyme of Chlamydomonas reinhardtii. In this study, the SAE1 and SAE2 partial sequences were successfully cloned by using specific primers respectively. The results showed that GST (glutathione S-transferase) can help the protein expression of SAE1 and SAE2 in Escherichia coli. The in vitro pull down assay did not reveal an interaction between SAE2 partial sequence and CrSUMO96-GG. Furthermore, I also used the GST-CrSAE2 partial sequence as the antigen for preparing the polyclonal antibody. Hopefully, it can be the specific tool for study of C. reinhardtii SUMO E1 activating enzyme in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:02:26Z (GMT). No. of bitstreams: 1 ntu-99-R97b47209-1.pdf: 10846793 bytes, checksum: 5c3bf9143a4fde484d684fb294498e0a (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要 i
Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 SUMO化修飾作用 1 1.1.1 SUMO 1 1.1.2 SUMO化修飾之反應機制 2 1.1.3 SUMO化修飾之生理功能 3 1.2 SUMO E1活化酶 4 1.2.1 SUMO E1活化酶之功能性區塊 4 1.2.2 SUMO E1活化酶催化之反應步驟 5 1.2.3 SUMO E1活化酶相關調控作用 6 1.3研究動機與方向 7 第二章 材料與方法 9 2.1實驗材料 9 2.1.1單胞綠藻 (Chlamydomonas reinhardtii) 9 2.2目標基因選殖 9 2.2.1 RNA之分離與純化 9 2.2.2反轉錄-聚合酶連鎖反應 (RT-PCR) 9 2.2.3聚合酶連鎖反應 (PCR) 10 2.3表現載體建構 11 2.3.1勝任細胞製備 11 2.3.2限制酶切反應 11 2.3.3接合反應 11 2.3.4質體轉形 (transformation) 12 2.4重組蛋白質之誘導表現 12 2.5重組蛋白純化方法 13 2.5.1 His6-tag重組蛋白質親和性層析法 13 2.5.2 GST-tag重組蛋白質親和性層析法 14 2.5.3蛋白質脫鹽與濃縮 14 2.6純化後重組蛋白分析 15 2.6.1 SDS膠體電泳 15 2.6.2蛋白質電泳轉印 16 2.6.3酵素免疫染色法 16 2.7 In vitro pull down assay 17 2.8多株抗體製備 17 2.8.1免疫注射 17 2.8.2小鼠尾尖採血 18 第三章 結果 19 3.1單胞綠藻SUMO E1活化酶之選殖 19 3.1.2 SAE1次單元體之選殖及定序結果分析 19 3.1.3 SAE2次單元體之選殖及定序結果分析 19 3.2單胞綠藻SUMO E1活化酶表現與純化 19 3.2.1 SAE1次單元體表現載體之建構 19 3.2.2 SAE1次單元體表現與純化 19 3.2.3 SAE2次單元體表現載體之建構 21 3.2.4 SAE2次單元體表現與純化 21 3.3 SAE1、SAE2部分序列與CrSUMO96-GG之in vitro pull down試驗 22 3.4 免疫染色測試SAE2次單元體多株抗體 23 第四章 討論 25 4.1以聚合酶連鎖反應選殖單胞綠藻SUMO E1活化酶 25 4.2選殖出之SAE1、SAE2次單元體序列與預測序列差異 26 4.3 SAE1、SAE2次單元體之表現與純化 27 4.4單胞綠藻SAE1、SAE2部分序列與CrSUMO96-GG之交互作用 28 4.5 SAE2多株抗體對CrSAE2序列之專一性辨認 29 參考文獻 31 圖與表 34 附錄 57 | |
dc.language.iso | zh-TW | |
dc.title | 單胞綠藻SUMO E1活化酶之基因選殖與蛋白質表現 | zh_TW |
dc.title | Molecular cloning and protein expression of SUMO E1
activating enzyme in Chlamydomonas reinhardtii | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊榮輝,張麗冠,陳威戎,吳俊宗 | |
dc.subject.keyword | 單胞綠藻,SUMO化修飾,E1活化酶,SAE1,SAE2, | zh_TW |
dc.subject.keyword | Chlamydomonas reinhardtii,sumoylation,E1 activating enzyme,SAE1,SAE2, | en |
dc.relation.page | 64 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2010-08-20 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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