請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64000
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立 | |
dc.contributor.author | Chao-Ming Hsieh | en |
dc.contributor.author | 謝詔名 | zh_TW |
dc.date.accessioned | 2021-06-16T17:26:01Z | - |
dc.date.available | 2017-09-19 | |
dc.date.copyright | 2012-09-19 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-16 | |
dc.identifier.citation | 1. J. J. Champoux, DNA topoisomerases: structure, function, and mechanism. Annual review of biochemistry 70, 369 (2001).
2. J. C. Wang, Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Quarterly reviews of biophysics 31, 107 (1998). 3. H. Y. Wu, S. H. Shyy, J. C. Wang, L. F. Liu, Transcription generates positively and negatively supercoiled domains in the template. Cell 53, 433 (1988). 4. R. A. Wasserman, C. A. Austin, L. M. Fisher, J. C. Wang, Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast. Cancer research 53, 3591 (1993). 5. K. Kirkegaard, G. Pflugfelder, J. C. Wang, The cleavage of DNA by type-I DNA topoisomerases. Cold Spring Harbor symposia on quantitative biology 49, 411 (1984). 6. C. Sissi, M. Palumbo, Effects of magnesium and related divalent metal ions in topoisomerase structure and function. Nucleic acids research 37, 702 (2009). 7. H. Hiasa, K. J. Marians, Topoisomerase III, but not topoisomerase I, can support nascent chain elongation during theta-type DNA replication. The Journal of biological chemistry 269, 32655 (1994). 8. R. A. Kim, J. C. Wang, Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase. The Journal of biological chemistry 267, 17178 (1992). 9. T. Z. Win, A. Goodwin, I. D. Hickson, C. J. Norbury, S. W. Wang, Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity. Journal of cell science 117, 4769 (2004). 10. A. Goodwin, S. W. Wang, T. Toda, C. Norbury, I. D. Hickson, Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe. Nucleic acids research 27, 4050 (1999). 11. S. Gangloff, B. de Massy, L. Arthur, R. Rothstein, F. Fabre, The essential role of yeast topoisomerase III in meiosis depends on recombination. The EMBO journal 18, 1701 (1999). 12. J. W. Wallis, G. Chrebet, G. Brodsky, M. Rolfe, R. Rothstein, A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell 58, 409 (1989). 13. J. L. Plank, S. H. Chu, J. R. Pohlhaus, T. Wilson-Sali, T. S. Hsieh, Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development. The Journal of biological chemistry 280, 3564 (2005). 14. J. Wu, L. Feng, T. S. Hsieh, Drosophila topo IIIalpha is required for the maintenance of mitochondrial genome and male germ-line stem cells. Proceedings of the National Academy of Sciences of the United States of America 107, 6228 (2010). 15. W. Li, J. C. Wang, Mammalian DNA topoisomerase IIIalpha is essential in early embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 95, 1010 (1998). 16. K. Y. Kwan, P. B. Moens, J. C. Wang, Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase III beta. Proceedings of the National Academy of Sciences of the United States of America 100, 2526 (2003). 17. K. Y. Kwan, J. C. Wang, Mice lacking DNA topoisomerase IIIbeta develop to maturity but show a reduced mean lifespan. Proceedings of the National Academy of Sciences of the United States of America 98, 5717 (2001). 18. P. Forterre, S. Gribaldo, D. Gadelle, M. C. Serre, Origin and evolution of DNA topoisomerases. Biochimie 89, 427 (2007). 19. L. Wu et al., The Bloom's syndrome gene product interacts with topoisomerase III. The Journal of biological chemistry 275, 9636 (2000). 20. P. Hu et al., Evidence for BLM and Topoisomerase IIIalpha interaction in genomic stability. Human molecular genetics 10, 1287 (2001). 21. R. J. Bennett, M. F. Noirot-Gros, J. C. Wang, Interaction between yeast sgs1 helicase and DNA topoisomerase III. The Journal of biological chemistry 275, 26898 (2000). 22. Y. Liu, S. C. West, More complexity to the Bloom's syndrome complex. Genes & development 22, 2737 (2008). 23. A. Ui et al., The ability of Sgs1 to interact with DNA topoisomerase III is essential for damage-induced recombination. DNA repair 4, 191 (2005). 24. C. Suski, K. J. Marians, Resolution of converging replication forks by RecQ and topoisomerase III. Molecular cell 30, 779 (2008). 25. R. J. Bennett, J. C. Wang, Association of yeast DNA topoisomerase III and Sgs1 DNA helicase: studies of fusion proteins. Proceedings of the National Academy of Sciences of the United States of America 98, 11108 (2001). 26. W. M. Fricke, V. Kaliraman, S. J. Brill, Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase. The Journal of biological chemistry 276, 8848 (2001). 27. S. Le Jan et al., Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis. Arteriosclerosis, thrombosis, and vascular biology, (2012). 28. R. Onodera et al., Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair. Genes & genetic systems 77, 11 (2002). 29. F. Fabre, A. Chan, W. D. Heyer, S. Gangloff, Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proceedings of the National Academy of Sciences of the United States of America 99, 16887 (2002). 30. G. Ira, A. Malkova, G. Liberi, M. Foiani, J. E. Haber, Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401 (2003). 31. K. Myung, A. Datta, C. Chen, R. D. Kolodner, SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination. Nature genetics 27, 113 (2001). 32. M. Wagner, G. Price, R. Rothstein, The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae. Genetics 174, 555 (2006). 33. J. Weinstein, R. Rothstein, The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1. DNA repair 7, 558 (2008). 34. E. Shor et al., Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae. Genetics 162, 647 (2002). 35. M. Azam et al., Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence. Nucleic acids research 34, 506 (2006). 36. L. Wu, I. D. Hickson, The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIalpha. Nucleic acids research 30, 4823 (2002). 37. C. Z. Bachrati, I. D. Hickson, Dissolution of double Holliday junctions by the concerted action of BLM and topoisomerase IIIalpha. Methods in molecular biology 582, 91 (2009). 38. J. L. Plank, J. Wu, T. S. Hsieh, Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration. Proceedings of the National Academy of Sciences of the United States of America 103, 11118 (2006). 39. K. Yamagata et al., Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases. Proceedings of the National Academy of Sciences of the United States of America 95, 8733 (1998). 40. L. Wu, I. D. Hickson, The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870 (2003). 41. M. Chang et al., RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex. The EMBO journal 24, 2024 (2005). 42. M. S. Lai, M. Seki, A. Ui, T. Enomoto, Rmi1, a member of the Sgs1-Top3 complex in budding yeast, contributes to sister chromatid cohesion. EMBO reports 8, 685 (2007). 43. J. R. Mullen, F. S. Nallaseth, Y. Q. Lan, C. E. Slagle, S. J. Brill, Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex. Molecular and cellular biology 25, 4476 (2005). 44. W. Bussen, S. Raynard, V. Busygina, A. K. Singh, P. Sung, Holliday junction processing activity of the BLM-Topo IIIalpha-BLAP75 complex. The Journal of biological chemistry 282, 31484 (2007). 45. F. Hartung, S. Suer, A. Knoll, R. Wurz-Wildersinn, H. Puchta, Topoisomerase 3alpha and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana. PLoS genetics 4, e1000285 (2008). 46. P. Cejka, J. L. Plank, C. Z. Bachrati, I. D. Hickson, S. C. Kowalczykowski, Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nature structural & molecular biology 17, 1377 (2010). 47. R. Hanai, P. R. Caron, J. C. Wang, Human TOP3: a single-copy gene encoding DNA topoisomerase III. Proceedings of the National Academy of Sciences of the United States of America 93, 3653 (1996). 48. E. Fritz, S. H. Elsea, P. I. Patel, M. S. Meyn, Overexpression of a truncated human topoisomerase III partially corrects multiple aspects of the ataxia-telangiectasia phenotype. Proceedings of the National Academy of Sciences of the United States of America 94, 4538 (1997). 49. J. Yang, C. Z. Bachrati, J. Ou, I. D. Hickson, G. W. Brown, Human topoisomerase IIIalpha is a single-stranded DNA decatenase that is stimulated by BLM and RMI1. The Journal of biological chemistry 285, 21426 (2010). 50. H. J. Tsai et al., Involvement of topoisomerase III in telomere-telomere recombination. The Journal of biological chemistry 281, 13717 (2006). 51. N. Temime-Smaali et al., Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres. The EMBO journal 27, 1513 (2008). 52. T. Wilson-Sali, T. S. Hsieh, Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIbeta, a type IA enzyme. The Journal of biological chemistry 277, 26865 (2002). 53. M. Arroyo, S. Bagchi, P. Raychaudhuri, Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex. Molecular and cellular biology 13, 6537 (1993). 54. S. Coulon et al., Slx1-Slx4 are subunits of a structure-specific endonuclease that maintains ribosomal DNA in fission yeast. Molecular biology of the cell 15, 71 (2004). 55. K. P. Hopfner et al., The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 418, 562 (2002). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64000 | - |
dc.description.abstract | DNA雙股螺旋結構的骨架由五碳糖與磷酸雙酯鍵所組成,因此具有彈性並可以進行小幅度的構形改變,故在DNA進行生理反應時,會因結構中出現的張力而導致超螺旋的生成,進而抑制反應的進行,或造成基因的不穩定。此外,DNA在進行修復或重組時,也會出現糾纏的中間物。上述這些DNA的拓樸問題 (DNA topological problems ),需要拓樸異構酶的活性才能順利解決。
拓樸異構酶在改變DNA的拓樸狀態時,會利用活性中心的酪胺酸,與DNA的磷酸鍵結形成phosphotyrosyl bond,進而打斷DNA。第一型拓樸異構酶打斷單股DNA,第二型則打斷雙股DNA。人類的拓樸異構酶三 (hTOPШ)在分類上屬於第一型的拓樸異構酶,且有Шα與Шβ兩種亞型,顯示在演化過程中TOPШ出現了功能分歧,兩者可能在生理功能上扮演不同的角色。目前已知hTOPШα會與BLM (RecQ-helicase family)及RMI1形成複合體,並利用TOPШα切斷與接合DNA的活性來解開與DNA重組之中間物Holliday Junction相似的DNA構型,可參與在DNA雙股斷裂與複製叉停滯的修復、端粒延長 (ALT),或是解開兩個複製叉會合時的DNA纏繞。因此,人類的TOPШα具有重要的生物功能。目前對TOPШβ的研究結果較為缺乏,只知道其與TOPШα差異在於不能與BLM以及RMI1形成複合體;在醫學上的研究則發現其活性可能與乳癌有關,但具體的機制卻仍不甚了解。 因此,本研究希望利用結構學的角度,來探討幾個問題:1. 人類TOPШα與TOPШβ功能上的差異為何? 2. 為什麼TOPШα可以和BLM及RMI1形成複合體,但TOPШβ卻不行? 3.與大腸桿菌的TOPШ序列比對後,發現人類TOPШ在C端多出了一段序列,其生理功能為何? 4. 解出人類TOPШα及TOPШβ的結構後,或可設計TOPШα的抑制劑,來阻斷癌細胞利用ALT 途徑進行端粒延長。本篇論文將對 hTOPШα及hTOPШβ蛋白表現量及可溶性的問題,進行改善。 | zh_TW |
dc.description.abstract | Cellular DNA exists in double-helical form as the most stable structure. Due to the intrinsic flexibility of its phosphodiester backbone, however, the DNA can undergo conformation change. In the presence of strains, usually caused by over- or under-winding of the base pairs, supercoils will be introduced that may inhibit DNA transactions or induce genome instability. Moreover, the repair of damaged DNA usually leads to intertwined intermediates. To maintain the topological homeostasis and genome integrity, many types of topoisomerases have evolved to solve various topological problems.
Mechanistically, topoisomerases use the active site tyrosine to cleave the phosphodiester DNA backbone by forming the phosphotyrosyl bond. Depending on whether cleavage occurs at one or both DNA strands, topoisomerases can be divided into type I and II, respectively. Human topoisomerase III (TOPIII) is classified as a type I toposiomerase, and possess two TOPIII isoforms – IIIα and IIIβ, suggesting that they may have specialized functions. Previous studies indicate that hTOPШα is a part of BLM core complex that also includes BLM (RecQ-helicase family) and RMI1 (RecQ-mediated genome instability protein 1). The DNA cleavage activity of hTOPIIIα is required for the resolution of Holliday Junction-like structures that occur during the repare of double strand break, stalled replication fork, alternative lengthing of chromosome pathway (ALT), and converging of replication forks. And the involvement of hTOPIIIα in ALT pathway suggests a potential anticancer therapeutic strategy. Unlike hTOPIIIα, hTOPIIIβ does not interact with BLM and RMI1. Though hTOPIIIβ is known to associate with M phase chromosomes, but its in vivo functions in human have remained poorly defined. The long term goal of this study is to understand the cellular functions of hTOPIIIα and hTOPIIIβ, and there are several outstanding questions to be addressed. 1. What are the functional differences between the two isoforms? 2. How can hTOPIIIα interact with BLM and RMI1 but hTOPIIIβ cannot? 3. Sequence alignment of hTOPIIIα and the E. coli TOP3 revealed that hTOPIIIα has an additional C-terminal domain, whose functional requires further structural and biochemical studies. 4. The structures of hTOPIIIα and hTOPIIIβ would be helpful for developing inhibitors that can be used to block the ALT pathway. We have constructed expression plasmids for both proteins, and the suitable conditions for expressing these two recombinant proteins have been extensively tested in E. coli. We are currently resolving the protein solubility issues as well as resort to yeast for protein expression. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:26:01Z (GMT). No. of bitstreams: 1 ntu-101-R99442029-1.pdf: 14667616 bytes, checksum: c24dd31eb52b0a0392b76719fb7c0c6f (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄
口試委員審定書.......................................................................................................................... II 謝誌............................................................................................................................................. III 中文摘要...................................................................................................................................... V 英文摘要..................................................................................................................................... VI 目錄.......................................................................................................................................... VIII 圖目錄........................................................................................................................................ XII 表目錄...................................................................................................................................... XIV 縮寫表....................................................................................................................................... XV 一、 前言............ ..........................................................................................................................1 1-1 拓樸異構酶的主要功能 .................................................................................................... 1 1-2 拓樸異構酶的分類與個別功能.......................................................................................... 2 1-3 真核生物之拓樸異構酶III (Topoisomerase III) ................................................................ 3 1-4 拓樸異構酶III與RecQ family DNA helicase的交互作用 ................................................ 4 1-5 拓樸異構酶III與Rmi1的交互作用 ................................................................................... 6 1-6 人類拓樸異構酶IIIα............................................................................................................ 7 1-7 人類拓樸異構酶IIIβ ........................................................................................................... 7 1-8 研究目的.............................................................................................................................. 8 二、 材料與方法......................................................................................................................... 9 2-1 蛋白表現質體之構築 ........................................................................................................ 9 2-1-1 pRH5-His10-hTOPIIIα(Δmit) , pET51b-hTOPIIIα(Δmit)-His6 ….......................................... 9 2-1-2 pET28a-His6-hTOPIIIβ.................................................................................................... 9 2-1-3 pET21b-His6-hTOPIIIα(30~654) ......................................................................................... 9 2-1-4 pMAL-c2F-MBP-hTOPIIIα(30-1001) -His6、pMAL-c2F-MBP-hTOPIIIα(30-654) -His6…. 13 2-1-5 pMAL-c2F-MBP-hTOPIIIβ(Y204C、S432P) -His6................................................................. 15 2-1-6 YepWob6-His6-hTOPIIIα(30-1001)..................................................................................... 16 2-1-7 YepWob6-His6-hTOPIIIα(30-654)...................................................................................... 16 2-1-8 YepWob6-His6-hTOPIIIα(759-1001) ................................................................................... 17 2-1-9 YepWob6-His6-hTOPIIIβ(1-863)....................................................................................... 17 2-1-10 pET41a-hTOPIIIα(Δmit)-His6、pCDFDue1-hRMI1-hRMI2-S....................................... 17 2-2 蛋白表現量的測試............................................................................................................ 18 2-2-1 pET21b-His6-hTOPIIIα(30~654)蛋白之表達.................................................................... 18 2-2-2 pMAL-c2F-MBP-hTOPIIIα(30-1001)-His6蛋白之表達…........…………..............…..… 20 2-2-3 pMAL-c2F-MBP-hTOPIIIα(30-654)-His6蛋白之表達..................................................... 20 2-2-4 pMAL-c2F-MBP-hTOPIIIβ(Y204C、S432P)-His6 蛋白之表達 ……….............................. 20 2-2-5 YepWob6-His6-hTOPIIIα(30-1001)蛋白之表達 (於YPG進行誘導)............................... 20 2-2-6 YepWob6-His6-hTOPIIIα(30-1001)蛋白之表達 (於SG-U進行誘導).............................. 24 2-2-7 YepWob6-His6-hTOPIIIα(30-1001)蛋白之表達 (於Raffinose以Galactose進行誘導)... 25 2-2-8 pET41a-hTOPIIIα(Δmit)-His6、pCDFDue1-hRMI1-hRMI2-S蛋白之共表達................. 26 2-3 蛋白純化............................................................................................................................ 26 2-3-1 pET21b-His6-hTOPIIIα(30~654)蛋白純化........................................................................ 26 2-3-2 pMAL-c2F-MBP-hTOPIIIβ(Y04C、S432P) -His6蛋白純化…………………................... 31 2-3-3 pET41a-hTOPIIIα(Δmit)-His6蛋白純化…………………............................................... 36 2-4 蛋白質濃縮與定量............................................................................................................ 36 2-5 蛋白質均質性測定............................................................................................................ 37 三、 結果.................................................................................................................................... 38 3-1 His6-hTOPIIIα(30~654)蛋白蛋白............................................................................................ 38 3-1-1構築pET21b-His6-hTOPIIIα(30~654) 表達質體…………………….....…..……..…….. 38 3-1-2 pET21b-His6-hTOPIIIα(30~654)蛋白表現………………..….....……..…..…………..... 38 3-1-3 pET21b-His6-hTOPIIIα(30~654)蛋白純化……………………...........................……..... 38 3-1-4 pET21b-His6-hTOPIIIα(30~654)蛋白純化效果測試 (使用non-ionic detergent – Igepal CA630進行萃取)……………………………………………………………..........… 39 3-1-5 pET21b-His6-hTOPIIIα(30~654)蛋白純化 (使用non-ionic detergent – Igepal CA630進行萃取 )……...............................................................................................................…. 39 3-1-6 pET21b-His6-hTOPIIIα(30~654)蛋白純化(使用non-ionic detergent –nonaethylene glycol monododecyl ether進行萃取 )..................................................................................... 40 3-2 MBP-hTOPIIIα 融合蛋白................................................................................................. 41 3-2-1 構築質體 pMAL-c2F-MBP-hTOPIIIα(30-1001) -His6 pMAL-c2F-MBP-hTOPIIIα(30-654) -His6 pMAL-c2F-MBP-hTOPIIIβ(Y204C、S432P) -His6.............................................. 41 3-2-2蛋白表現 pMAL-c2F-MBP-hTOPIIIα(30-1001) -His6 pMAL-c2F-MBP-hTOPIIIα(30-654) -His6 pMAL-c2F-MBP-hTOPIIIβ(Y204C、S432P) -His6...........................................…. 42 3-2-3 pMAL-c2F-MBP-hTOPIIIβ(Y204C、S432P) -His6蛋白純化.............................................. 42 3-3 使用酵母菌表達系統表現蛋白........................................................................................ 43 3-3-1 構築質體 YepWob6-His6-hTOPIIIα(30-1001) YepWob6-His6-hTOPIIIα(30-654) YepWob6-His6-hTOPIIIα(759-1001) YepWob6-His6-hTOPIIIβ.......................................................................... 43 3-3-2蛋白表現 YepWob6-His6-hTOPIIIα(30-1001) YepWob6-His6-hTOPIIIα(30-654) YepWob6-His6-hTOPIIIα(759-1001) YepWob6-His6-hTOPIIIβ............................................................................ 44 3-4 hTOPIIIα(Δmit)與hRMI1之共表達...................................................................................... 44 3-4-1 pET41a-hTOPIIIα(Δmit)-His6、pCDFDue1-hRMI1-hRMI2-S蛋白之共表達.............. 44 3-4-2 pET41a-hTOPIIIα(Δmit)-His6蛋白純化........................................................................ 45 四、 討論..................................................................................................................................... 46 4-1 pET21b-hTOPIIIα(30~654)蛋白.............................................................................................. 46 4-2 pET21b-hTOPIIIα(30~654)蛋白 ( 使用non-ionic detergent進行萃取)............................... 47 4-3 MBP融合蛋白………………..………………………………………...........………....... 48 4-4 於酵母菌中表現蛋白........................................................................................................ 51 4-5 hTOPIIIα(Δmit)與hRMI1之共表達................................................................................... 52 4-6 pET41a-hTOPIIIα(Δmit)-His6蛋白純化............................................................................. 53 4-7 結論.................................................................................................................................... 53 圖……………………………............…………………………………………………………. 55 表.. …………………………………............………………………………………………….. 85 參考文獻……………………………………............………………………………………..... 90 圖目錄 圖 2-1 pMAL-c2F-hTOPIIIβ(Y204C、S432P)蛋白二級結構分析……………....…...………..… 55 圖3-1 pET21b-hTOPIIIα(30~654)小量表現測試……………… …………………..……….… 56 圖3-2 hTOPIIIα(30~654)鎳離子親和性管柱純化……………………...……………..…….… 57 圖 3-3 hTOPIIIα(30~654)之heparin管柱純化………………………………...………………. 58 圖 3-4 hTOPIIIα(30~654)之分子篩管柱純化……………………………………………..…... 59 圖 3-5 hTOPIIIα(30~654)之蛋白表現量測試…………………………………………...…….. 60 圖 3-6 hTOPIIIα(30~654)使用non-ionic detergent Igepal CA630萃取結果 …………...…….61 圖 3-7 hTOPIIIα(30~654)使用Igepal CA630萃取之鎳離子親和性管柱純化…………….… 62 圖 3-8 hTOPIIIα(30~654)使用Igepal CA630萃取之heparin管柱純化……………………… 63 圖 3-9 hTOPIIIα(30~654)使用Igepal CA630萃取及heparin管柱純化之西方墨點法分析… 64 圖 3-10 hTOPIIIα(30~654)使用nonaethylene glycol monododecyl ether萃取之鎳離子親和性管柱純化……………………………………………………………………………. 65 圖 3-11 hTOPIIIα(30~654)使用nonaethylene glycol monododecyl ether萃取之heparin管柱純化……………………………………………………………………………..……... 66 圖 3-12 hTOPIIIα(30~654)使用nonaethylene glycol monododecyl ether萃取之Q管柱純化……………………………………………………………………………….….. 67 圖 3-13 hTOPIIIα(30~654)使用nonaethylene glycol monododecyl ether萃取純化結果分析…………………………………………………………………...…...……..…... 68 圖 3-14 MBP 融合蛋白小量表現測試....……………………….………………………… 69 圖 3-15 hTOPIIIβ(Y204C、S432P) MBP融合蛋白之amylose管柱純化..…………………......... 70 圖 3-16 hTOPIIIβ(Y204C、S432P) MBP融合蛋白使用factor Xa進行MBP切割之環境測試………………………………………………………….…………….……….. 71 圖 3-17 hTOPIIIβ(Y204C、S432P)分子篩管柱純化……………….…………………………..… 72 圖 3-18 hTOPIIIβ(Y204C、S432P) 融合蛋白之鎳離子親和性管柱純化……….......................... 73 圖 3-19 hTOPIIIβ(Y204C、S432P) 融合蛋白之heparin管柱純化.……………..………….……. 74 圖 3-20 hTOPIIIβ(Y204C、S432P) 融合蛋白之分子篩管柱純化………………………….......... 75 圖 3-21 DLS分析結果圖…………………….…………..…………………………………. 76 圖 3-22 於酵母菌系統的小量表現測試 – 於YPG誘導 (2-2-5方法)............................. 77 圖 3-23 於酵母菌系統的小量表現測試 - 於SD-U誘導 (2-2-6方法)............................ 78 圖 3-24 於酵母菌系統的小量表現測試 – 於Rffinose使用galactose誘導 (2-2-7方法). 79 圖 3-25 於酵母菌系統的小量表現測試 –– 於YPG誘導 (2-2-5方法)........................... 80 圖 3-26 pET41a-hTOPIIIα(Δmit)-His6、pCDFDue1-hRMI1-hRMI2-S蛋白之共表達................ 81 圖 3-27 pET41a-hTOPIIIα(Δmit)-His6之鎳離子親和性管柱純化........................................... 82 圖 3-28 pET41a-hTOPIIIα(Δmit)-His6之分子篩管柱純化....................................................... 83 圖 4-1 TOPIII之序列比較…………………………………………………………………...84 表目錄 表2-1 本實驗使用的菌種............................................................................................ 85 表2-2 本實驗使用的質體............................................................................................ 86 表2-3 藥品配置............................................................................................................ 87 | |
dc.language.iso | zh-TW | |
dc.title | 人類拓樸異構酶Шα及Шβ之純化與功能解析 | zh_TW |
dc.title | Toward Structural Studies of Human Topoisomerase Шα and Topoisomerase Шβ | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 孔繁璐,徐駿森 | |
dc.subject.keyword | 拓樸異構酶,IIIα,拓樸異構酶,IIIβ,BLM,RMI1,端粒延長, | zh_TW |
dc.subject.keyword | Topoisomerase IIIα,Topoisomerase IIIβ,BLM,RMI1,ALT pathway, | en |
dc.relation.page | 94 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 14.32 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。