臺灣博碩士論文加值系統

本論文分為兩部份，第一部分為檸檬蛋白質雙硫鍵異構酶之基因選殖、蛋白質表現及酵素特性分析。第二部份為樟芝麩胱甘肽轉移酶之蛋白質表現及酵素特性分析。
蛋白質雙硫鍵異構酶對雙硫鍵具有氧化、還原之功能，因而可幫助蛋白質形成新的雙硫鍵及促進蛋白質重新摺疊。利用聚合酶鏈鎖反應，從檸檬選殖出蛋白質雙硫鍵異構酶之全長 cDNA，轉譯區包含1503 bp、500個胺基酸，分子量為61.5 kD。發現具有兩個活性區位,分別位於胺基酸60-63、405-408，具有氧化還原的功能。以 pET-20b(+) 作為表現載體，轉形到 E. coli Rosetta ( DE3 ) pLysS 作表現，再利用含鎳螯合物凝膠之親和性管柱來純化，最後利用 scrambled RNase A 測試蛋白質雙硫鍵異構酶之活性，其 KM、Vmax 和 Kcat 的值分別為 0.015 nM、0.35 nM h -1 和 1.06 x 10-4 h-1。
麩胱甘肽轉移酶屬於第二型代謝解毒酶，高度表現於人類肝臟和肺臟組織中，其主要催化還原態麩胱甘肽與環氧化合物結合，以達到保護個體對抗細胞毒素和致癌物質之傷害。樟芝麩胱甘肽轉移酶之全長 cDNA 包含651 bp 轉譯區、216 個胺基酸，分子量為25.0 kD。以 pYEX-S1 作為表現載體，轉形到 S.cerevisiae CK3，再利用含鎳螯合物凝膠之親和性管柱來純化，最後利用1-氯-2,4-二硝基苯來測試樟芝麩胱甘肽轉移酶之活性，其 KM、Vmax 和 Kcat 的值分別為8.49 mM、0.39 nMh-1和13.03 h-1。

目錄

謝辭 I
摘要 II
Abstract IV
縮寫表 IX
壹、前言 1
Part I 檸檬蛋白質雙硫鍵異構酶 1
一、檸檬簡介 1
二、 蛋白質雙硫鍵異構酶 ( protein disulfide isomerase，PDI ) 2
三、蛋白質雙硫鍵異構酶體系結構 ( domain architecture ) 5
四、人類蛋白質雙硫鍵異構酶家族 ( human PDI family ) 6
Part II 樟芝麩胱甘肽轉移酶 7
一、樟芝簡介 7
二、麩胱甘肽 ( glutathione, GSH ) 8
三、麩胱甘肽轉移酶 ( glutathione S-transferase, GST ) 10
貳、實驗材料 12
一、材料 12
(一) 材料來源 12
(二) 載體 ( vector ) 12
(三) 宿主 ( host ) 12
二、培養基 ( medium ) 13
(一) Escherichia coli 13
(二) yeast 13
三、儀器設備 14
四、實驗套組 15
五、實驗藥品試劑 15
(一) 酵素 ( enzyme ) 15
(二) 標準品 ( marker ) 15
(三) 染劑 15
(四) 緩衝液 16
(五)培養基添加物 16
(六)蛋白質電泳相關藥品 16
(七)純化相關藥品 16
(八)活性測試相關物品 16
(九)其他 17
參、實驗方法 19
一、檸檬mRNA之萃取 19
二、cDNA之合成 21
三、檸檬之基因選殖 23
(一) 檸檬蛋白質雙硫鍵異構酶 ( ClPDI ) 之基因比對 23
(二) 選殖檸檬蛋白質雙硫鍵異構酶基因 23
(三) 瓊脂膠體電泳 ( agarose gel electrophoresis ) 24
(四) 瓊脂膠體回收與目標 DNA 純化 ( gel elution ) 25
(五) 選殖載體之建構與 cDNA 片段序列確認 26
(六) 檸檬蛋白質雙硫鍵異構酶之全長分析 27
四、檸檬蛋白質雙硫鍵異構酶與樟芝麩胱甘肽轉移酶基因之建構( construction ) 28
(一) 基因全長之選殖 28
(二) 抽取質體 DNA ( plasmid extraction ) 28
(三) 限制酶切割 29
(四) 勝任細胞之製備 30
(五) 重組質體與轉形作用 30
五、檸檬蛋白質雙硫鍵異構酶與樟芝麩胱甘肽轉移酶重組蛋白質之表現與純化 31
(一) 誘導蛋白質之表現 31
(二) 蛋白質之純化 32
(三) SDS-聚丙烯醯胺凝膠電泳( sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE ) 33
(四) 西方墨點轉漬 ( western blotting ) 36
(五) 蛋白質濃縮透析與濃度測定 38
(六) 蛋白質濃度測定 38
六、活性測定 ( activity assay ) 39
(一) 蛋白質雙硫鍵異構酶活性測定之反應式及原理 39
(二) 麩胱甘肽轉移酶活性測定之反應式及原理 41
七、酵素特性分析 ( enzyme characterization ) 42
(一) 熱穩定性 42
(二) pH 值 42
(三) imidazole 43
(四) DTT 43
肆、結果與討論 44
Part I 檸檬蛋白質雙硫鍵異構酶 44
一、檸檬蛋白質雙硫鍵異構酶之基因選殖 44
二、檸檬蛋白質雙硫鍵異構酶重組蛋白質之表現與純化 47
三、檸檬蛋白質雙硫鍵異構酶家族歸類 49
四、檸檬蛋白質雙硫鍵異構酶之活性測試 49
五、檸檬蛋白質雙硫鍵異構酶之特性分析 51
Part II 樟芝麩胱甘肽轉移酶 53
一、樟芝麩胱甘肽轉移酶重組蛋白質之表現與純化 53
二、樟芝麩胱甘肽轉移酶之酵素動力 55
三、樟芝麩胱甘肽轉移酶之特性分析 56
伍、附圖 57
Figure 1. Agarose gel analysis. 58
Figure 2. DNA and amino acid sequence of ClPDI. 59
Figure 3. Alignment of the amino acid sequence of PDI with other organisms and 3-D homology structure. 60
Figure 4. Purification of the recombinant ClPDI. 61
Figure 5. Western blot demonstration of the presence of ClPDI in Rosetta ( DE3 ) pLysS. 61
Figure 6. Western blots of TcGST and AnPDIA. 62
Figure 7. The activity of PDI was assayed. 63
Figure 8. The standard curve using different concentrations of RNase A to RNA in 5 min. 63
Figure 9. The initial rate of the enzymatic reaction was measured with the varied sRNase A concentration. 64
Figure 10. Double-reciprocal plot of ClPDI activity at varying concentrations of sRNase A. 64
Figure 11. Effect of temperature, pH, imidazole, DTT on the purified ClPDI. 65
Figure 12. Alignment of the amino acid sequence of GST with other organisms and 3-D homology structure. 66
Figure 13. Purification of the recombinant TcGST. 67
Figure 14. Molecular mass of TcGST was determined via ESI-Q-TOF mass spectrometry(Micromass, Manchester, England). The molecular mass obtained was 14304 Da. 68
Figure 15. The initial rate of the enzymatic reaction was measured with the varied CDNB concentration. 69
Figure 16. Double-reciprocal plot of TcGST activity at varying concentrations of CDNB. 69
Figure 17. Effect of temperature, pH, imidazole on the purified TcGST. 70
陸、References 71
柒、Appendix 76
Appendix 1. Citrus limonum. 77
Appendix 2. Protein disulfide isomerase and thioredoxin expression in response to hypersaline stress. 78
Appendix 3. ER lumen and calreticulin(CRT)/calnexin (CNX) cycle. 79
Appendix 4. Molecular interactions in antigen presentation and host responses to virus infections. 80
Appendix 5. PDI contains an active-site with two reduced cysteine sulfhydryl (–SH) groups. 81
Appendix 7. The human PDI family. 83
Appendix 8. Taiwanofungus camphoratus. 84
Appendix 9. pCR2.1-TOPO vector. 85
Appendix 10. pCR4-TOPO vector. 86
Appendix 11. pET-20b(+) vector. 87
Appendix 12. Map and cloning site of pYEX-S1. 88
Appendix 13. Amino Acid Sequence of Bovine Ribonuclease. 89
Appendix 14. Reestablishing correct disulfide pairing. 90
Appendix 15. GST assay. 91
Appendix 16. DTNB only assay. 91
Figure 17. GST active sites. 92
Figure 18. Mammalian GST structures. 92

1. Miyake,C., Asada K. 1992. Thylakoid-bound ascorbate perxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids.Plant Cell Physiology, 33, 541-553
2. Sliskovic I., Raturi A., Mutus B. 2005. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem ; 280:8733-8741.
3. Cuozzo, J., Kaiser C. 1999. Competition between glutathione and protein thiolsfor disulphide-bond formation. Nature Cell Biol 1: 130-135.
4. Frand, A., Kaiser C. 1998. The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum. Mol Cell 1: 161-170.
5. Tu, B., Ho-Schleyer, S., Travers, K. 2000. Biochemical basis of oxidative protein folding in the endoplasmic reticulum. Science 290: 1571-1574.
6. Kleizen, B., Braakman, I. 2004. Protein folding and quality control in the endoplasmic reticulum. Curr Opin Cell Biol 16, 343-349.
7. Malhotra, J., Kaufman, R. 2007. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 18, 716-731.
8. Zhang, K., Kaufman, R. 2006. Protein folding in the endoplasmic reticulum and the unfolded protein response. Handb Exp Pharmacol, 69-91.
9. Wajih, N., Hutson S., Wallin R. 2007. Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction. J Biol Chem 282:2626-2635.
10. Wells, W., Xu D., Yang Y., 1990. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem 265:15361-15364.
11. Janiszewski, M., Lopes L., Carmo A., 2005. Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells. J Biol Chem 280:40813-40819.
12. Appenzeller-Herzog, C., Ellgaard, L. 2008. The human PDI family: versatility packed into a single fold. Biochim Biophys Acta 1783:535-548.
13. Lee, A. S. 2001. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26:504-510.
14. Jordan, P. A., Gibbins J. M. 2006. Extracellular disulfide exchange and the regulation of cellular function. Antioxid Redox Signal 8:312-324.
15. Ou, W., J. Silver. 2006. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion. Virology 350:406-417.
16. Weisbart, R. H. 1992. An antibody that binds a neutrophil membrane protein, ERp72, primes human neutrophils for enhanced oxidative metabolism in response to formyl-methionyl-leucyl-phenylalanine. Implications for ERp72 in the signal transduction pathway for neutrophil priming. J Immunol 148:3958-3963.
17. Turano, C., Coppari, S., Altieri, F. 2002. Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell Physiol 193:154-163.
18. Ni, M., Lee, A. 2007. ER chaperones in mammalian development and human diseases. FEBS Lett 581, 3641-3651.
19. Turano, C., Coppari, S., Altieri, F. 2002. Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell Physiol 193:154-163.
20. Farquhar, R., Honey, N., Murant, S.J. 1991. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae. Gene 108, 81-89.
21. LaMantia, M., Miura, T., Tachikawa, H. 1991. Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast. Proc Natl Acad Sci U S A 88, 4453-4457.
22. Tachikawa, H., Miura, T., Katakura, Y., Mizunaga, T. 1991. Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth. J Biochem 110, 306-313.
23. Lovat, P.E., Corazzari, M., Armstrong, J.L. 2008. Increasing melanoma cell death using inhibitors of protein disulfide isomerases to abrogate survival responses to endoplasmic reticulum stress. Cancer Res 68, 5363-5369.
24. Severino, A., Campioni, M., Straino, S. 2007. Identification of protein disulfide isomerase as a cardiomyocyte survival factor in ischemic cardiomyopathy. J Am Coll Cardiol 50, 1029-1037.
25. Shan, S.W., Tang, M.K., Cai, D.Q. 2005. Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death. Dev Dyn 233, 266-281.
26. Gumireddy, K., Sun, F., Klein-Szanto, A.J. 2007. In vivo selection for metastasis promoting genes in the mouse. Proc Natl Acad Sci U S A 104, 6696-6701.
27. Annamari P. 2003. The significance of the domains of protein disulfide isomerase for the different functions of the protein Oulu University Library http://herkules.oulu.fi/isbn9514271726/html/index.html
28. Koivu, J., Helaakoski, T., Pihlajaniemi, T. 1987. A single polypeptide acts both as the β subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase. J Biol Chem 262: 6447-6449.
29. Pihlajaniemi, T., Helaakoski, T., Tasanen, K. 1987. Molecular cloning of the β -subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene. EMBO J 6: 643-649.
30. Wetterau, J., Combs, K., Spinner, S. 1990. Protein disulfide isomerase is a component of the microsomal triglyseride transfer protein complex. J Biol Chem 265: 9801-9807.
31. Cai, H., Wang, C., Tsou, C. 1994. Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds. J Biol Chem 269: 24550-24552.
32. Song, J., Wang, C. 1995. Chaperone-like activity of protein disulfide isomerase in the refolding of rhodanase. Eur J Biochem 231: 312-316.
33. Wang, C., Tsou, C. 1993. Protein disulfide isomerase is both an enzyme and a chaperone. FASEB J 7: 1515-1517.
34. Yao, Y., Zhou, Y., Wang, C. 1997. Both the isomerase and chaperone activities of protein disulphide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2. EMBO J 16: 651-658.
35. Kersteen, E., Raines, R. 2003. Catalysis of protein folding by protein disulfide isomerase and small-molecule mimics. Antioxid Redox Signal 5, 413-424.
36. Wu, S., Ryvarden, L.,Chang, T. 1997. Antrodia camphorata (“niuchang-chih”), new combination of a medicinal fungus in Taiwan. Bot.Bull. Acad. Sin. 38, 273-275.
37. Freedman, R., Hirst, T., Tuite, M. 1994. Protein disulphide-isomerase: building bridges in protein folding. Trends Biochem Sci 19: 331-336.
38. Houston, S., Ray, S., Chitsike, I. 2005. Breaking the silence: an HIV-related educational intervention for medical students in Zimbabwe. Cent Afr J Med. 51(5-6): 48-52.
39. Yao, Y., Zhou, Y., Wang ,C. 1997. Both the isomerase and chaperone activities of protein disulphide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2. EMBO J 16: 651-658.
40. Goldberger, R., Epstein, C., Anfinsen, C. 1963. Acceleration of reactivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver. J Biol Chem 238: 628-635.
41. 王伯徹、黃仁彰. 靈芝與樟芝之研發與是場面面觀. 食品工業2002, 349(5): 3-17.
42. Sliskovic, I., Raturi, A., Mutus, B. 2005. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem ; 280:8733-8741.
43. Lee, I., Huang, R., Chen, C. 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects.FEMS Microbiology letters , 209:63-67.
44. Meister, A. 1988. Glutathione metabolism and its selective modification. J. Biol. Chem. 263:17205-17208.
45. Keppler, D. 1999. Export pumps for glutathione S-conjugates. Free Rad. Biol. Med. 27:985-991
46. Kensler, T., Groopman J., Sutter T. 1999. Development of cancer chenopreventive agents: Oltipraz as a paradigm. Chem. Res. Toxicol. 12:113-126.
47. Sheehan, D., Meade, G., Foley V. 2001. Structure, function and evolution of glutathione transferase: Implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360: 1-16.
48. Eaton, D., Bammler T. 1999. Concise review of glutathione S-transferase and their significance to toxicology. Toxicol. Sci. 49:156-164.
49. Ni, M., Lee, A. 2007. ER chaperones in mammalian development and human diseases. FEBS Lett 581, 3641-3651.
50. Sheehan, D., Meade, G., Foley V. 2001. Structure, function and evolution of glutathione transferase: Implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360: 1-16.
51. Celina, N., Jeenes, D., Punt P. 2000. Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway of Aspergillus niger. Appl. Environ. Microbiol. 66(2):775-782.
52. Ferrari, D., Soling, H. 1999. The protein disulphide-isomerase family :unravelling a string of folds. Biochem. J. 339:1-10.
53. Sliskovic, I., Raturi, A., Mutus B. 2005. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem ; 280:8733-8741.
54. Wang, C., Tsou, C. 1993. Protein disulfide isomerase is both an enzyme and a chaperone. FASEB J 7: 1515-1517.
55. Yao, Y., Zhou, Y., Wang ,C. 1997. Both the isomerase and chaperone activities of protein disulphide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2. EMBO J 16: 651-658.
56. Sliskovic, I., Raturi, A., Mutus B. 2005. Characterization of the S-denitrosation activity of protein disulfide isomerase. J Biol Chem ; 280:8733-8741.
57. Song, J., Wang, C. 1995. Chaperone-like activity of protein disulfide isomerase in the refolding of rhodanase. Eur J Biochem 231: 312-316.
58. Houston, S., Ray, S., Chitsike, I. 2005. Breaking the silence: an HIV-related educational intervention for medical students in Zimbabwe. Cent Afr J Med. 51(5-6): 48-52.
59. Song, T., Yen, G. 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J. Agirc. Food Chem. 50:3322-3327.
60. Tachikawa, H., Miura, T., Katakura, Y. 1991. Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth. J Biochem 110, 306-313.
61. Tsai, Z. T., Liaw, S., L.1985. The use and the effect of Ganoderma .San Yun Press: Taichung , Taiwan , 116.
62. Dirr, H., Reinemer, P., Huber, R. 1994. X-ray crystal structures of cytosolic glutathione S-transferase: Implications for protein architecture, substrate recognition and catalytic function. Eur. J. Biochem. 220:645-661.
63. 廖怡珍. 樟芝 2-Cys peroxiredoxin 之基因選殖及其特性分析. 國立臺灣海洋大學 生物科技研究所 碩士學位論文, 2006.
64. 姜郁琦. 甘藷去氫抗壞血酸還原酶與樟芝穀氧還蛋白之基因選殖、表現及酵素特性分析. 國立臺灣海洋大學 生物科技研究所 碩士學位論文, 2008.
65. 陳政仁. 甘藷穀胱甘胺酸還原酶之基因選殖、表現及酵素特性分析與樟芝穀胱甘胺酸還原酶之基因選殖. 國立臺灣海洋大學 生物科技研究所 碩士學位論文, 2009.
66. 郭恬岎. Pdia4基因初步分析及其基因剔除小鼠的建立. 國立臺灣海洋大學 水產養殖學系碩士學位論文, 2010.
67. 鍾佑瑜. 利用慢病毒載體研究蛋白質雙硫異構酶在T細胞的功能. 國立臺灣海洋大學 生物科技研究所 碩士學位論文, 2009.