臺灣博碩士論文加值系統

穀胱甘肽依賴型甲醛脫氫酶 (glutathione-dependent formaldehyde dehydrogenase；GFD或GSH-FDH) 於去除甲醛毒性或抗氧化上扮演很重要的角色。本實驗以樟芝 (Antrodia camphorata) 子實體為研究材料，建立其cDNA庫，依據其他物種GFD之胺基酸序列保守區域設計簡併性引子，選殖出樟芝GFD之cDNA序列。其轉譯區共可轉譯出378個胺基酸，與其他物種GFD之胺基酸序列比對具有高度保守性。為分析樟芝GFD酵素活性，將其轉譯區接合至表現載體pET-20b(+)，並轉形宿主大腸桿菌表現，續以鎳螯合親和性管柱層析純化重組GFD蛋白質。純化之酵素於10% SDS-PAGE上可看出單一明顯色帶。其酵素活性於50℃加熱5 min仍保有約50%活性，且安定於pH 8至11之鹼性環境，經由凝乳胰蛋白酶處理40 min仍不影響GFD酵素活性。此酵素具有雙重功能，除GFD活性外，亦有亞硝醯穀胱甘肽還原酶 (S-nitrosoglutathione reductase；GSNOR) 活性，被認為可保護細胞抵抗亞硝化壓力。利用GFD及GSNOR活性分析方法對GFD受質氫氧甲基穀胱甘肽 (S-hydroxymethylglutathione；HMGSH) 及亞硝醯穀胱甘肽(S-nitrosoglutathione；GSNO) 進行酵素動力實驗，求得KM為1.20 mM及0.28 mM。為研究GFD之活性區域，進一步將樟芝GFD轉譯區及其第47號半胱胺酸突變絲氨酸 (Cys47 to Ser47；C47S) 之突變型構築至pYEX-S1載體及轉形至酵母菌宿主表現，經由鎳螯合親和性管柱層析純化樟芝GFD及C47S，C47S已無GFD活性，但GSNOR活性仍剩16%。此符合Cys47於酵素催化上扮演重要角色。經樟芝GFD轉形之酵母菌CK3可以抵抗甲醛誘導之細胞死亡，其於0.36 µmol/cm2甲醛存在下仍有60%的存活率，但C47S突變型則無。進一步將樟芝GFD基因進行密碼子最優化並構築至pcDNA3，再轉染人類胚胎腎293T細胞。優化之樟芝GFD於293T細胞中不僅比野生型具有較高表現量，且具有較高能力以抵抗GSNO所誘導之細胞死亡。利用DETA/NOy作為NO來源處理細胞，樟芝GFD並無法保護細胞抵抗DETA/NO誘導的細胞死亡。因此樟芝具有很強的專一性來進行抵抗GSNO所誘導之細胞死亡。

Glutathione-dependent formaldehyde dehydrogenase (GFD or GSH-FDH) plays important roles in formaldehyde detoxification and antioxidation. A gene encoding GFD from Antrodia camphorata was identified based on sequence homology. The deduced amino acid sequence of 378 amino acid residues is conserved among the reported GFDs. To characterise the Ac-GFD, the coding region was subcloned into a vector pET-20b(+) and transformed into Escherichia coli. The recombinant GFD was expressed and purified by Ni2+-nitrilotriacetic acid Sepharose. This purified enzyme showed a single band on a 10% SDS-PAGE. The enzyme retained 50% GFD activity after heating at 50℃ for 5 min. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) presumably to safeguard against nitrosative stress. The KM values for S-hydroxymethylglutathione and S-nitrosoglutathione were 1.20 and 0.28 mM, respectively. To investigate the active motif of Ac-GFD, both the coding region of Ac-GFD and its C47S mutant were subcloned into a vector pYEX-S1 and transformed into yeast, both the Ac-GFD and C47S were purified by Ni2+-nitrilotriacetic acid Sepharose, the C47S mutant lost GFD activity, but still remained 16% GSNOR activity. This behavior conformed that Cys47 plays an important role in enzymatic activity. The CK3 yeast transformed with Ac-GFD showed 60% viability against formaldehyde-induced cell death under 0.36 µmol/cm2 but not for C47S mutant. We further introduced the codon optimization of Ac-GFD into a vector pcDNA3 and transfected into 293T cells. The optimized Ac-GFD in 293T cells was not only much higher expression than wild type but also higher ability against GSNO-induced cell death. Using DETA/NO as a NO donor, the Ac-GFD can’t protect the cell against the DETA/NO- induced cell death. This suggested Ac-GFD against GSNO-induced cell death is highly specific.

謝誌 I
摘要 II
Abstract IV
第一章 前言 1
1.1 臺灣特有真菌牛樟芝 1
1.2 穀胱甘肽 2
1.3 甲醛 3
1.4 穀胱甘肽依賴型甲醛脫氫酶 4
1.5 一氧化氮 5
1.6 亞硝醯硫化物及亞硝醯穀胱甘肽 7
1.7 氧化/亞硝化壓力對蛋白質硫基的修飾 7
1.7.1自由基之定義 7
1.7.2 氧化壓力與亞硝化壓力 8
1.7.3 蛋白質硫基亞硝醯化作用 10
1.7.4 蛋白質硫基穀胱甘肽化作用 11
1.8自由基與中樞神經退化性疾病 11
1.9穀胱甘肽與細胞凋亡之關係 13
1.10 研究動機 15
第二章 材料與方法 17
2.1 實驗設計 17
2.2 實驗材料 18
2.2.1 研究物種 18
2.2.2 載體 18
2.2.3 宿主 18
2.2.4 引子 19
2.2.5 藥品試劑 20
2.2.6 試劑套組 26
2.2.7 儀器設備 27
2.3 實驗方法 28
2.3.1 樟芝GFD基因之選殖 28
2.3.1.1 mRNA之抽取 28
2.3.1.2 cDNA library之製備 30
2.3.1.3 GFD簡併性引子設計 30
2.3.1.4 聚合酶鏈鎖反應 31
2.3.1.5 DNA/RNA電泳分析 31
2.3.1.6 選殖載體接合反應 32
2.3.1.7 選殖載體轉形選殖宿主 32
2.3.1.8 菌落PCR篩選及基因定序 33
2.3.1.9 5’端Ac-GFD cDNA選殖 33
2.3.1.10 軟體分析cDNA全長 33
2.3.2 重組基因表現載體之構築與轉形表現宿主 34
2.3.2.1 限制酶切位引子設計 34
2.3.2.2 膠體回收與目標DNA萃取 35
2.3.2.3 質體DNA之抽取 35
2.3.2.4 限制酶切割作用 36
2.3.2.5 表現載體之處理製備 37
2.3.2.6 目標DNA與表現載體之接合 37
2.3.2.7 重組質體轉形及序列確認 38
2.3.2.8 勝任細胞之製備 39
2.3.2.9 重組質體與表現宿主之轉形作用 39
2.3.2.10 酵母菌表現宿主之轉形作用 39
2.3.3 Ac-GFD重組蛋白質之表現及純化 40
2.3.3.1 重組蛋白質於E. coli之誘導表現 40
2.3.3.2 重組蛋白質於yeast之表現 40
2.3.3.3 Ni-NTA親和性管柱層析純化重組蛋白質 41
2.3.3.4 利用聚丙烯醯胺膠體電泳進行蛋白質分析 42
2.3.3.5 重組蛋白質透析及保存 43
2.3.4 Ac-GFD重組蛋白質之酵素活性測定與分析 44
2.3.4.1 BradFord法之蛋白質含量測定 44
2.3.4.2 GFD & GSNOR 活性分析 44
2.3.4.3 GFD與GSNOR活性染色 45
2.3.4.4 酵素動力學分析 46
2.3.4.5 ESI Q-TOF MS測定分子量 47
2.3.4.6 Ac-GFD特性分析 47
2.3.5 定點突變分析 49
2.3.5.1 定點突變Ac-GFD基因 49
2.3.5.2 活性分析 50
2.3.6 酵母菌培養試驗 50
2.3.7 細胞培養 50
2.3.7.1 細胞解凍 50
2.3.7.2 細胞收集與冷凍保存 51
2.3.7.3 細胞計數 51
2.3.7.4 細胞過渡性轉染 51
2.3.7.5 細胞lysate製備 52
2.3.8 Ac-GFD表現於人類細胞之密碼子利用率最優化 52
2.3.9 細胞處理分析測試 53
2.3.9.1 GSNO處理細胞 53
2.3.9.2 MTT分析方法測試細胞存活率 54
2.3.9.3 西方點墨法分析蛋白質表現量 55
2.3.9.4 AnnexinV-FITC細胞凋亡試驗 56
2.3.9.5 DETA/NO 處理試驗 56
第三章 結果 58
3.1 樟芝GFD之cDNA全長選殖 58
3.2 Ac-GFD蛋白質3-D結構預測 59
3.3 重組Ac-GFD於大腸桿菌之表現及純化 60
3.4 重組Ac-GFD之酵素動力試驗 61
3.5 重組Ac-GFD之生化特性分析 61
3.6 重組Ac-GFD與C47S突變株於酵母菌表現之活性 62
3.7 GSNO對細胞存活影響 64
3.8 Ac-GFD轉染細胞之表現及其對GSNO之影響 65
3.9 Ac-GFD表現於人類細胞之密碼子利用率最優化 66
3.10 C47S突變區對細胞抵抗GSNO能力影響 67
3.11 GSNO誘導細胞凋亡試驗 68
3.12 DETA/NO做為NO來源測試細胞 69
第四章 討論與展望 70
第五章 參考文獻 77
圖表 84
Table 1. Kinetic characterisation of Ac-GFD and other published ADH3. 84
Figure 1. Agarose gel electrophoresis analysis of Ac-GFD genes amplified by PCR. 85
Figure 2. cDNA sequence encoding the amino acid of Ac-GFD. 86
Figure 3. Optimal alignment of the amino acid sequences of GFD from various sources and homology structure prediction. 87
Figure 4. Agarose gel electrophoresis analysis of constructed Ac-GFD genes. 89
Figure 5. Coomassie Brilliant Blue R-250-stained SDS–polyacrylamide gel showing the purification of His6-tagged Ac-GFD expressed in E. coli. 90
Figure 6. Molecular mass determination by ESI Q-TOF MS. 91
Figure 7. Double-reciprocal plots of varying HMGSH (A), NAD+(B), GSNO (C), and NADH (D) on Ac-GFD activity. 92
Figure 8. Effect of temperature on the purified Ac-GFD. 93
Figure 9. Effects of pH, SDS, imidazole and chymotrypsin on the purified Ac-GFD. 94
Figure 10. Ac-GFD possessing GSNOR activity on gel. 95
Figure 11. Coomassie Brilliant Blue R-250-stained SDS– polyacrylamide gel showing the purification of His6-tagged Ac-GFD expressed in yeast. 96
Figure 12. Enzyme activity of recombinant Ac-GFD purified from E. coli or yeast expression. 97
Figure 13. Ac-GFD purified from yeast possessed alchol dehydrogenase activity. 98
Figure 14. Coomassie Brilliant Blue R-250-stained SDS– polyacrylamide gel showing the purification of His6-tagged C47S expressed in yeast. 99
Figure 15. Amino acid Cysteine 47 to Serine mutant of Ac-GFD shows enzyme activities decreased. 100
Figure 16. Colony formation of Ac-GFD and C47S mutant against formaldehyde. 101
Figure 17. 293T viabilities during GSNO incubation. 102
Figure 18. Transfection efficiency of Ac-GFD into 293T cells and its expression. 103
Figure 19. 293T cells transfected with Ac-GFD against GSNO-induced cell death. 104
Figure 20. Expression of the Ac-GFD in 293T cells. 105
Figure 21. Nucleotide sequences of Ac-GFD cDNA and codon usage optimization. 106
Figure 22. Ac-GFD-O was expressed in 293T cells showing less aggregation. 107
Figure 23. Codon optimization of Ac-GFD into 293T cells improved cell viability. 108
Figure 24. Ac-GFD expression in 293T cells is improved by codon optimization. 109
Figure 25. Effect of GFD and codon optimization of GFD on GSNO-induced 293T cell viability and target gene expression. 110
Figure 26. Transfection efficiency of GFD in 293T cells. 111
Figure 27. Effect of GFD on GSNO-induced 293T cell viability. 112
Figure 28. Effect of GFD on GSNO reduction. 113
Figure 29. Effect of GFD on GSNO-induced embryonic kidney cells apoptosis. 114
Figure 30. Effect of GFD on DETA/NO-induced cell viability. 115
附錄 116
Appendix 1.1 Antrodia camphorata (bolotype). 116
Appendix 1.2 Scheme for ADH class III isozyme operating as a formaldehyde dehydrogenase and the chemical structure of GSNO. 117
Appendix 1.3 Scheme for the ADH class III isozyme catabolism of GSNO included GSH. 118
Appendix 1.4 Chemical relationship among different reactive oxygen species (ROS) and reactive nitrogen species (RNS), and their impact on some post-translational modification of proteins. 119
Appendix 1.5 Probable reactions of protein S-glutathionylation. 120
Appendix 1.6 Apoptotic signaling pathways. 121
Appendix 1.7 Molecular mechanisms involved in the regulation of apoptosis by GSH. 122
Appendix 2.1 Map of pCR®4-TOPO®. 123
Appendix 2.2 Map of pET-20b(+). 124
Appendix 2.3 Map of pYEX-S1. 125
Appendix 2.4 Map of pcDNA3. 126
Appendix 2.5 Map of pEGFP-N2. 127
Appendix 2.6 Map of pUC57. 128
Appendix 3.1 Nucleotide sequence of 3’ partial Ac-GFD cDNA ligated with pCR®4-TOPO®. 129
Appendix 3.1 Nucleotide sequence of 3’ partial Ac-GFD cDNA ligated with pCR®4-TOPO®. 130
Appendix 3.2 Nucleotide sequence of 5’ partial Ac-GFD cDNA ligated with pCR®4-TOPO®. 131
Appendix 3.3 Nucleotide sequence of pETGFD recombinant DNA. 132
Appendix 3.4 Nucleotide sequence of pYGFD recombinant DNA. 133
Appendix 3.5 Nucleotide sequence of pYC47S recombinant DNA. 134
Appendix 3.6 Nucleotide sequence of pcGFD recombinant DNA. 135
Appendix 3.7 Nucleotide sequence of pcEGFP recombinant DNA. 136
Appendix 3.8 Nucleotide sequence of pcGFDG recombinant DNA. 137
Appendix 3.9 Nucleotide sequence of pcGFD-O recombinant DNA. 138
Appendix 3.10 Nucleotide sequence of pcGFD-OG recombinant DNA. 139
Appendix 3.11 Nucleotide sequence of pcC47S-O recombinant DNA. 140
著作 141

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