請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60562
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 樓國隆(Kuo-Long Lou) | |
dc.contributor.author | Dung-Chi Wu | en |
dc.contributor.author | 吳東祈 | zh_TW |
dc.date.accessioned | 2021-06-16T10:21:43Z | - |
dc.date.available | 2018-09-24 | |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-16 | |
dc.identifier.citation | 1. Huang, H.C., et al., Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. J Biol Chem, 2003. 278(47): p. 46750-9.
2. Shi, C.S., et al., Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation, 2005. 111(13): p. 1627-36. 3. Esmon, C.T., Protein C. Prog Hemost Thromb, 1984. 7: p. 25-54. 4. Esmon, C.T., Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J, 1995. 9(10): p. 946-55. 5. Suzuki, K., et al., Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J, 1987. 6(7): p. 1891-7. 6. Conway, E.M., Thrombomodulin and its role in inflammation. Semin Immunopathol, 2012. 34(1): p. 107-25. 7. Dodd, R.B. and K. Drickamer, Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. Glycobiology, 2001. 11(5): p. 71R-9R. 8. Villoutreix, B.O. and B. Dahlback, Molecular model for the C-type lectin domain of human thrombomodulin. Journal of Molecular Modeling, 1998. 4(10): p. 310-322. 9. Weisel, J.W., et al., The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy. J Biol Chem, 1996. 271(49): p. 31485-90. 10. Conway, E.M., et al., The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. J Exp Med, 2002. 196(5): p. 565-77. 11. Li, Y.H., et al., The role of thrombomodulin lectin-like domain in inflammation. J Biomed Sci, 2012. 19: p. 34. 12. Scaffidi, P., T. Misteli, and M.E. Bianchi, Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature, 2002. 418(6894): p. 191-5. 13. Gardella, S., et al., The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep, 2002. 3(10): p. 995-1001. 14. Abeyama, K., et al., The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest, 2005. 115(5): p. 1267-74. 15. Shi, C.S., et al., Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response. Blood, 2008. 112(9): p. 3661-70. 16. Wouters, M.A., et al., Evolution of distinct EGF domains with specific functions. Protein Sci, 2005. 14(4): p. 1091-103. 17. Tolkatchev, D. and F. Ni, Calcium binding properties of an epidermal growth factor-like domain from human thrombomodulin. Biochemistry, 1998. 37(25): p. 9091-100. 18. Tohda, G., et al., Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol, 1998. 18(12): p. 1861-9. 19. Hamada, H., et al., The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood, 1995. 86(1): p. 225-33. 20. Kurosawa, S., et al., A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site. J Biol Chem, 1988. 263(13): p. 5993-6. 21. Zushi, M., et al., The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem, 1989. 264(18): p. 10351-3. 22. Suzuki, K., et al., A domain composed of epidermal growth factor-like structures of human thrombomodulin is essential for thrombin binding and for protein C activation. J Biol Chem, 1989. 264(9): p. 4872-6. 23. Wang, W., et al., Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem, 2000. 275(30): p. 22942-7. 24. Schenk-Braat, E.A., J. Morser, and D.C. Rijken, Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator. Eur J Biochem, 2001. 268(21): p. 5562-9. 25. Honda, G., et al., The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function. J Biochem, 1995. 118(5): p. 1030-6. 26. Bourin, M.C., E. Lundgren-Akerlund, and U. Lindahl, Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem, 1990. 265(26): p. 15424-31. 27. Koyama, T., et al., Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III. Eur J Biochem, 1991. 198(3): p. 563-70. 28. Tsiang, M., S.R. Lentz, and J.E. Sadler, Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. J Biol Chem, 1992. 267(9): p. 6164-70. 29. Conway, E.M., B. Nowakowski, and M. Steiner-Mosonyi, Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. J Cell Physiol, 1994. 158(2): p. 285-98. 30. Huntington, J.A., Structural Insights into the Life History of Thrombin, in Recent Advances in Thrombosis and Hemostasis 2008, Kenzo Tanaka, et al., Editors. 2008, Springer Japan: Tokyo. p. pp. 80-106. 31. Lane, D.A., H. Philippou, and J.A. Huntington, Directing thrombin. Blood, 2005. 106(8): p. 2605-12. 32. Doyle, M.F. and K.G. Mann, Multiple active forms of thrombin. IV. Relative activities of meizothrombins. J Biol Chem, 1990. 265(18): p. 10693-701. 33. Bode, W., et al., The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J, 1989. 8(11): p. 3467-75. 34. Schechter, I. and A. Berger, On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun, 1967. 27(2): p. 157-62. 35. Davie, E.W. and J.D. Kulman, An overview of the structure and function of thrombin. Semin Thromb Hemost, 2006. 32 Suppl 1: p. 3-15. 36. Harris, J.L., et al., Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A, 2000. 97(14): p. 7754-9. 37. Ohkubo, S., et al., Substrate phage as a tool to identify novel substrate sequences of proteases. Comb Chem High Throughput Screen, 2001. 4(7): p. 573-83. 38. Petrassi, H.M., et al., A strategy to profile prime and non-prime proteolytic substrate specificity. Bioorg Med Chem Lett, 2005. 15(12): p. 3162-6. 39. Huntington, J.A., Molecular recognition mechanisms of thrombin. J Thromb Haemost, 2005. 3(8): p. 1861-72. 40. Krishnaswamy, S., Exosite-driven substrate specificity and function in coagulation. J Thromb Haemost, 2005. 3(1): p. 54-67. 41. Huntington, J.A., How Na+ activates thrombin--a review of the functional and structural data. Biol Chem, 2008. 389(8): p. 1025-35. 42. Pirkle, H., et al., On the location in the thrombin B chain of substrate recognition sites for fibrinopeptide release and factor XIII activation. Thromb Res, 1989. 55(6): p. 737-46. 43. De Cristofaro, R., et al., The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding. FEBS J, 2006. 273(1): p. 159-69. 44. Carter, I.S., et al., Thrombin a-chain: activation remnant or allosteric effector? Thrombosis, 2010. 2010: p. 416167. 45. Monroe, D.M. and M. Hoffman, What does it take to make the perfect clot? Arterioscler Thromb Vasc Biol, 2006. 26(1): p. 41-8. 46. Crawley, J.T., et al., The central role of thrombin in hemostasis. J Thromb Haemost, 2007. 5 Suppl 1: p. 95-101. 47. Mosesson, M.W., Fibrinogen and fibrin structure and functions. J Thromb Haemost, 2005. 3(8): p. 1894-904. 48. Mann, K.G., K. Brummel, and S. Butenas, What is all that thrombin for? J Thromb Haemost, 2003. 1(7): p. 1504-14. 49. Marguerie, G.A., E.F. Plow, and T.S. Edgington, Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem, 1979. 254(12): p. 5357-63. 50. De Candia, E., et al., Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem, 2001. 276(7): p. 4692-8. 51. Esmon, C.T., Molecular events that control the protein C anticoagulant pathway. Thromb Haemost, 1993. 70(1): p. 29-35. 52. Vindigni, A., et al., Energetics of thrombin-thrombomodulin interaction. Biochemistry, 1997. 36(22): p. 6674-81. 53. Fuentes-Prior, P., et al., Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex. Nature, 2000. 404(6777): p. 518-25. 54. Andersson, H.M., et al., Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain. Blood, 2010. 115(23): p. 4878-85. 55. Griffin, J.H., B.V. Zlokovic, and L.O. Mosnier, Protein C anticoagulant and cytoprotective pathways. Int J Hematol, 2012. 95(4): p. 333-45. 56. Schneider, I., Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol, 1972. 27(2): p. 353-65. 57. Santos, M.G., et al., Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology, 2007. 54(1): p. 15-24. 58. Brillet, K., C.A. Pereira, and R. Wagner, Expression of membrane proteins in Drosophila Melanogaster S2 cells: Production and analysis of a EGFP-fused G protein-coupled receptor as a model. Methods Mol Biol, 2010. 601: p. 119-33. 59. Dmitri, I.S. and H.J.K. Michel, Small-angle scattering studies of biological macromolecules in solution. Reports on Progress in Physics, 2003. 66(10): p. 1735. 60. Svergun, D., Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography, 1992. 25(4): p. 495-503. 61. Mathews, II, et al., Structure of a nonadecapeptide of the fifth EGF domain of thrombomodulin complexed with thrombin. Biochemistry, 1994. 33(46): p. 13547-52. 62. Srinivasan, J., et al., Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects. Biochemistry, 1994. 33(46): p. 13553-60. 63. Adler, M., et al., The structure of a 19-residue fragment from the C-loop of the fourth epidermal growth factor-like domain of thrombomodulin. J Biol Chem, 1995. 270(40): p. 23366-72. 64. Sampoli Benitez, B.A., et al., Structure of the fifth EGF-like domain of thrombomodulin: An EGF-like domain with a novel disulfide-bonding pattern. J Mol Biol, 1997. 273(4): p. 913-26. 65. Wood, M.J., B.A. Sampoli Benitez, and E.A. Komives, Solution structure of the smallest cofactor-active fragment of thrombomodulin. Nat Struct Biol, 2000. 7(3): p. 200-4. 66. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970. 227(5259): p. 680-5. 67. DiBella, E.E., M.C. Maurer, and H.A. Scheraga, Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J Biol Chem, 1995. 270(1): p. 163-9. 68. Aretz, S., et al., In-depth mass spectrometric mapping of the human vitreous proteome. Proteome Sci, 2013. 11(1): p. 22. 69. Konarev, P.V., et al., PRIMUS: a Windows PC-based system for small-angle scattering data analysis. Journal of Applied Crystallography, 2003. 36(5): p. 1277-1282. 70. Konarev, P.V., et al., PRIMUS: a Windows PC-based system for small-angle scattering data analysis. Journal of Applied Crystallography, 2003. 36: p. 1277-1282. 71. Svergun, D.I., Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J, 1999. 76(6): p. 2879-86. 72. Franke, D. and D.I. Svergun, DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. Journal of Applied Crystallography, 2009. 42(2): p. 342-346. 73. Volkov, V.V. and D.I. Svergun, Uniqueness of ab initio shape determination in small-angle scattering. Journal of Applied Crystallography, 2003. 36(3 Part 1): p. 860-864. 74. Wriggers, W., Conventions and workflows for using Situs. Acta Crystallographica Section D, 2012. 68(4): p. 344-351. 75. Petoukhov, M.V. and D.I. Svergun, Global Rigid Body Modeling of Macromolecular Complexes against Small-Angle Scattering Data. Biophysical journal, 2005. 89(2): p. 1237-1250. 76. Vandenborre, G., et al., Glycosylation signatures in Drosophila: fishing with lectins. J Proteome Res, 2010. 9(6): p. 3235-42. 77. Budzynski, A.Z., Chromogenic Substrates in Coagulation and Fibrinolytic Assays. Lab Medicine, 2001. 32(7): p. 365-368. 78. Izquierdo, C. and F.J. Burguillo, Synthetic substrates for thrombin. Int J Biochem, 1989. 21(6): p. 579-92. 79. Dang, Q.D. and E. Di Cera, Chromogenic substrates selective for activated protein C. Blood, 1997. 89(6): p. 2220-2. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60562 | - |
dc.description.abstract | 人類凝血酶調節素(Thrombomodulin; TM)是一大小約為70kDa的I型跨膜糖蛋白,主要表現在於上皮細胞表面。人類TM基因無內含子,其轉譯出的蛋白質含多個結構域,從N-端開始,為一類C型凝集素結構區段(Lectin-C like domain; TMD1),主要和發炎反應相關,其後則是接了6個類EGF重複序列(6x EGF-like repeats; TMD2),TM即是透過這6個類EGF重複與凝血酶(thrombin)或是和其他蛋白質如蛋白質C (protein C)或凝血酶啟動纖溶抑制物(thrombin-activatable fibrinolysis inhibitor)相互作用,再來則是一段絲氨酸/蘇氨酸豐富的區域(Serine/Threonine-rich domain; TMD3),其上有四個潛在的O-連接糖基化位點,最後則是一個跨膜段(TMD4)和位於細胞質中的短尾巴(TMD5)。
TM在生理上表現了多種功能:包含血液凝結(coagulation),血纖維蛋白溶解(fibrinolysis),發炎反應(inflammation),以及細胞粘附與增生(cell adhesion and proliferation)。但是,TM的最主要功能則是作為輔助因子與凝血酶 (thrombin)形成複合物,與TM的結合會幫助凝血酶誘導抗凝途徑中蛋白質C (protein C) 活化,而這可提高thrombin活化protein C的速度將近千倍。與TM在功能上的發現的重要性相比,目前對於TM的結構的研究,無論是結構域或是整體,都是相對有限的,目前唯一解出的晶體結構是TM 的一個片段,EGF4-6,與thrombin形成的複合物(Thrombin-Thrombomodulin EGF4-6 complex)。而為了了解TM是藉由何種機制來增強thrombin-induced的 protein C activation達到千倍,這可能需要對TM的結構有更進一步的了解才有辦法解釋,因此我們便想試著表現不同結構區段的TM來對其進行結構的研究。 從先前的數據,大腸桿菌與酵母菌似乎都不是一個適合表現TM的系統。 先前TMD1(只有Lectin-C like domain)在大腸桿菌中的表達,結果會形成包涵體。然而在酵母菌表現系統中的結果,再次發現有蛋白質聚集的情形。所以,這一次我們決定使用不同的系統來表現TM的各個區段。最後我們選擇的是果蠅細胞表現系統,(Drosophila Schneider 2 cell; S2 cells)。 S2 cells表現系統的優點是,昆蟲細胞的醣基化修飾較酵母菌單純,並且也容易培養。同時我們為了探討TM是藉由何種機制來調節凝血酶活化蛋白質C,我們也試著進一步利用S2 cells來表現蛋白質C以及凝血酶。 在本篇論文中,我們成功地克隆到TM的不同結構域區段,包括了Lectin-C domain(LC; TMD1),EG-like domain(EGF; TMD2),以及soluble TM(sTM; TMD12)。同時我們也克隆了wild-type/mutant thrombin。將這些成功構築的質體轉染進S2 cells中,並且利用hygromycin B來做篩選,直到stable clone篩選完成後才開始進行重組蛋白的表現。我們利用ELISA和LC-MS/MS來確認表現的重組蛋白,同時也利用酵素活性分析來驗證表現的重組蛋白確實具有生理活性,而這些結果表明,利用S2細胞作為蛋白質表現系統是適合於我們的研究標的。而在本篇論文中,我們嘗試利用目前已經日趨成熟的小角度X光散射(SAXS)實驗技術來研究不同區段的TM結構。在小角度X光散射實驗中,我們利用 ATSAS 程式分析 TMD12的蛋白分子數據以及TMD2在添加鈣離子以及不添加鈣離子的情形下的蛋白分子數據,並利用 DAMMIF 及 Situs 重建蛋白表面構造(ab-initio envelope)。同源模型(homology model)的構築則是利用Swiss-model database進行同源模擬來模擬TMD1的結構,而TMD2的EGF1-3亦同樣利用Swissmodel database,利用TMD2已解出的EGF4-6晶體結構來模擬,並將這些模擬與小角度X光散射實驗所得之表面構造進行比較,並且反計算出同源模擬之小角度X光散射曲線後與實際實驗數值比較,結果顯示同源模型與重建之表面構造符合。 | zh_TW |
dc.description.abstract | Thrombomodulin (TM) is a 70 kDa type I transmembrane glycoprotein mainly expressed on the epithelial cell surface. The intron-less gene of human TM encoded a multiple domains protein including an N-terminal C-type lectin-like domain (TMD1) which may associate in inflammation, six EGF-like repeats (TMD2) which would interact with thrombin and the other proteins such as protein C or thrombin-activatable fibrinolysis inhibitor, a serine/threonine-rich region (TMD3) which have four potential O-linked glycosylation sites, a single transmembrane segment (TMD4) and a short cytoplasmic tail (TMD5).
TM exhibits a range of physiologically multiple functions: coagulation, fibrinolysis, inflammation, cell adhesion, and cell proliferation. But the major function of TM is as a cofactor in the thrombin-induced activation of protein C in the anticoagulant pathway by forming a complex with thrombin, and this would raises the speed of protein C activation thousand-fold. In spite of its importance, structural studies of TM and its domains are limited; only crystal structures of a fragment of TM EGF4-6 complex with thrombin are available. In order to understand the mechanism of how TM enhances thrombin-induced protein C activation by thousand-fold, besides, there are several counteracting molecules complexed with thrombomodulin and fine-tuning the haemostatsis, such as thrombin-thrombomodulin-protein C may turn the coagulation cascade off, thrombin-thrombomodulin-TAFI which would shut-down fibrinolysis cascade. These dynamic mechanisms of how thrombomodulin modulate thrombin are unclear and remained to be resolved. To date, the only structure been resolved for thrombomodulin is EGF domain 4-6. In this study, we aim for expressing TM for functional and structural studies. From our previous data, yeast or E. coli seemed not be suitable for expressing TM. The expression of TMD1, consisting of only the C-type lectin-like domain, in E. coli results in the formation of inclusion bodies. And TMD1 expressed in yeast show again the protein aggregation. So, this time we decide to use the different system and plasmid to express the various domains of TM. We want to use the Drosophila Schneider 2 cell (S2 cell) line expression system. The advantage of S2 cell system is that the insect cell has relatively simple glycosylation to yeast, and it is also easily cultivated. And to explore how the EGF-like domains of TM modulate thrombin activity on protein C activation, we also aimed to express protein C and thrombin in S2 cell for further study. In this study, we successfully cloned the lectin-C like domain (LC; TMD1), EGF-like domain (EGF; TMD2), and soluble form of TM (sTM; TMD12) for S2 cell expression. We also cloned the wild-type/mutant thrombin. The stable transfected clones have been selected by using hygromycin B. In the study, we successfully expressed pre-thrombin-2, Protein C and TM recombinant proteins in S2 cells analyzed by LC-MS/MS. Further, we also demonstrated that activated thrombin by ecarin indeed activates Protein C in the presence of TM. The activation activity is about thousand fold by ELISA. Then, we take advantage of the currently matured small-angle X-ray scattering (SAXS) technique to investigate the TM structure. In the SAXS experiment, we collected the SAXS data of TMD12, TMD2 with calcium and TMD2 without calcium, which have been characterized by programs implant in ATSAS, ab-initio envelope of each protein was reconstructed by DAMMIF and Situs. And the homology models of TMD12 and TMD2 have been built, TMD1 were built from swissmodel database according to sequence alignment by Swissmodel database and TMD2 were built by the crystal structure of resolved TM EGF4-6. And these models have been docked into average envelops and assessed. The results showed that the homology model can dock into SAXS envelope pretty well. And we found that EGF domain of TM may induce some conformational change by calcium. This is the first time, even though it is just an envelope, to view the conformation and calcium effect of EGF like domain 1~6 of TM structurally. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:21:43Z (GMT). No. of bitstreams: 1 ntu-102-R00442023-1.pdf: 8625836 bytes, checksum: a413219ac7ff58a5cece2f60b583f28e (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | CONTENTS
口試委員會審定書 誌謝 i 中文摘要 ii ABSTRACT v CONTENTS viii LIST OF TABLES xi LIST OF FIGURES xii LIST OF ABBREVIATION xiv Chapter 1 Introduction 1 1.1 Thrombomodulin 1 1.1.1 The function and structure of thrombomodulin 1 1.1.2 C-type lectin-like domain 1 1.1.3 Epidermal Growth Factor-like (EGF-like) domain 2 1.1.4 Serine/Threonine-rich domain 3 1.1.5 Transmembrane domain and cytoplasmic domain 4 1.2 Thrombin 4 1.2.1 Chemistry of thrombin 4 1.2.2 Thrombin in haemostasis 6 1.3 Drosophila expression system 7 1.4 Small-Angle X-ray Scattering 8 1.5 The goal of this study 10 Chapter 2 Material and methods 12 2.1 Material 12 2.1.1 Plasmid 12 2.1.2 Cell line 12 2.1.3 Reagent 12 2.1.4 Broth, plate and medium of E. coli incubation 13 2.1.5 Medium, buffer and reagents of cell culture 14 2.1.6 Column chromatography 14 2.1.7 Reagents for column chromatography 14 2.1.8 SDS-PAGE 15 2.2 Preparation of recombinant human thrombomodulin 16 2.2.1 Construction of the expression plasmid 16 2.2.2 Cell culture and transfection 17 2.2.3 Cell line selection 18 2.2.4 Purification of secreted recombinant human thrombomodulin 18 2.2.5 Detect the expression of recombinant human thrombomodulin by SDS-PAGE and western blotting 19 2.2.6 ELISA, enzyme-linked immunosorbent assay 20 2.3 Preparation of recombinant a-thrombin 21 2.3.1 Construction of the expression plasmid 21 2.3.2 Cell culture and transfection 21 2.3.3 Cell line selection 21 2.3.4 Purification of secreted recombinant prethrombin-2 22 2.3.5 Detect the expression of recombinant prethrombin-2 by SDS-PAGE and western blotting 22 2.3.6 Activation of recombinant prethrombin-2 22 2.3.7 ELISA, enzyme-linked immunosorbent assay 23 2.4 Protein Assay 24 2.5 Functional analysis of activated a-thrombin and thrombomodulin 24 2.6 LC-Mass/Mass spectrometry sample preparation 25 2.7 Small angle X-ray scattering (SAXS) experiment 26 Chapter 3 Results 28 3.1 Protein production 28 3.1.1 Thrombomodulin 28 3.1.2 Prethrombin-2 29 3.2 Analysis of expressed recombinant protein 31 3.2.1 Analysis of expressed recombinant protein by SDS-PAGE 31 3.2.2 Identification of recombinant protein by ELISA 32 3.2.3 Identification of recombinant protein by LC-MS/MS 32 3.2.4 Functional analysis of recombinant protein 33 3.3 Structural analysis by Small-angle X-ray scatteing 35 3.3.1 SAXS sample preparation 35 3.3.2 SAXS data analysis 35 3.3.3 Ab initio envelope reconstruction and protein model docking 37 3.3.4 Homology rigid-body modeling 38 Chapter 4 Discussion 40 Chapter 5 Reference 41 Chapter 6 Tables 46 Chapter 7 Figures 48 | |
dc.language.iso | en | |
dc.title | 人類凝血酶調節素不同區段之表現、純化以及分析 | zh_TW |
dc.title | Expression, Purification and Structural Analysis of Various Domains of Human Thrombomodulin | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 李玉梅(Yu-May Lee),沈三泰(San-Tai Shen) | |
dc.contributor.oralexamcommittee | 廖彥銓(Yen-Chywan Liaw),游偉絢(Wei-Hsuan Yu) | |
dc.subject.keyword | 凝血酶,調節素,類EGF重複序列,鈣離子,小角度X光散射, | zh_TW |
dc.subject.keyword | Thrombomodulin,EGF-like repeats,Calcium,Small-angle X-ray Scattering(SAXS), | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-16 | |
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-102-1.pdf 目前未授權公開取用 | 8.42 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。