臺灣博碩士論文加值系統

本研究利用大腸杆菌大量生產重組差向異構酶，會在胞內形成大量的包涵體(inclusion bodies)，故發展出一個最適的差向異構酶包涵體復性程序，從包涵體回復成具有天然活性的蛋白質。先以探討最適的溶解條件，找出pH 12.5 ,100mM Tris-Buffer和0.3 % SDS 具有高溶解效應。並以pH12.5,50mM Tris-Buffer當做最適溶解劑，得到最佳的溶解蛋白濃度8mg/ml，再以HCl將溶解蛋白質溶液調控至中性進行復性再摺疊程序，可知調控至pH8有最佳的可溶蛋白回收率，並發現形成而大量沒有活性的寡聚體(oligomer)，並試圖以溫度、NaCl、添加劑、及氧化系統來改善回收率和再摺疊後無活性之情形。
且本研究應用氧化改組系統(oxido-shuffling system)，添加GSH和GSSH來使雙硫鍵正確配對。可得復性差向異構酶二聚體之相對比活性為30%。

GlcNAc 2-epimerase was expressed in Escherichia coli in the form of inlcusion bodies. The epimerase from the purified inclusion bodies was solubilized in 100 mM Tris-HCl buffer at pH 12.5 and > 0.3 % SDS had significatly solubilization effect. The optimal conditions for denaturation of epimerase were when inclusion bodies were solubilized in 50mM Tris-HCl buffer. The refolding method is that the solubilized protein was adjusted to neutral pH. Optimal soluble protein recovery is about 65% when the solubilized proteins (8mg/ml) were refolded at pH 8 .In this study,we added GSH and GSSG to promoted activity of refolded epimerase utilizing an oxido-shuffling system .The dimer fractions of refolded epimerase had 30% relative specific activity .

中文摘要 I
目錄 III
圖目錄 VII
表目錄 VIII
第一章、 緒論 1
1-1 前言 1
1-2實驗動機與目的 2
第二章 文獻回顧與原理 3
2-1 蛋白質的穩定與摺疊 3
2-2包涵體的形成 4
2-2-1 包涵體的回收、純化 5
2-2-2包涵體的溶解 6
2-3蛋白質復性 10
2-3-1蛋白質復性方式 10
2-3-2雙硫鍵(disufide-bond)的形成 12
2-3-3摺疊與集結之間的動力學競爭: 13
2-4 增加復性產率 14
2-4-1蛋白質復性環境的最適化 14
2-4-2添加化學輔助劑 14
2-4-3 復性方式的改變 18
2-5 N-乙醯胺D-葡萄糖2-差向異構酶介紹 25
第三章 實驗藥品與儀器 27
3-1實驗藥品 27
3-2 實驗儀器 28
第四章 實驗方法 30
4-1 菌種的培養 30
4-1-1 LB固態洋菜培養基的製備 30
4-1-2 LB液態培養基的製備 30
4-1-3菌種培養方式 31
4-1-4 菌種醱酵槽培養 31
4-2菌體、包涵體、活性蛋白質的回收、純化 31
4-2-2 包涵體的回收 32
4-2-3 包涵體的純化 32
4-2-4可溶活性差向異構酶的純化 33
4-3包涵體溶解 34
4-3-1溶解劑對包涵體溶解之探討 34
4-3-2 pH值對包涵體溶解之探討 34
4-3-3 SDS濃度對包涵體溶解之探討 35
4-3-4 最適包涵體溶解量之探討 35
4-4 差向異構酶之復性 35
4-4-1 pH與差向異構酶濃度對復性之探討 36
4-4-2 溫度效應對差向異構酶復性之探討 36
4-4-3 NaCl對差向異構酶復性之探討 36
4-5添加劑對差向異構酶復性之探討 37
4-5-1 Arginine對差向異構酶復性之探討 37
4-5-2 PEG對差向異構酶復性之探討 37
4-6差向異構酶氧化復性 38
4-6-1 GSH與GSSG對差向異構酶復性之探討 38
4-6-2 GSH與GSSG對差向異構酶階段復性之探討 38
4-7包涵體溶解時間對差向異構酶復性之探討 39
4-8 實驗分析方法 39
4-8-1差向異構酶活性分析 39
4-8-2 差向異構酶濃度分析 41
4-8-3 大小排除管柱層析分析 41
4-8-4 SDS-PAGE電泳分析 42
第五章 結果與討論 44
5-1 差向異構酶之表現 44
5-2包涵體之溶解 44
5-2-1溶解劑對包涵體溶解效應 44
5-2-2 pH值對包涵體溶解之影響 45
5-2-3 SDS對包涵體溶解之影響 46
5-2-4最適包涵體溶解度 46
5-3 差向異構酶之復性 47
5-3-1 pH與差向異構酶濃度對復性之影響 47
5-3-2 溫度對差向異構酶復性之影響 48
5-3-3 NaCl對差向異構酶復性之探討 49
5-4添加劑對差向異構酶復性之探討 50
5-4-1 L-Arginine對差向異構酶復性之影響 50
5-4-2 PEG對差向異構酶復性之影響 50
5-5差向異構酶氧化復性 51
5-5-1 GSH與GSSG對差向異構酶復性之探討 51
5-5-2 GSH與GSSG對差向異構酶階段復性之探討 52
第六章 結論 53
第七章 參考文獻 54
附錄一 79
附錄二 80
附錄三 81
附錄四 82
附錄五 83
附錄六 84
圖目錄
圖2-4-1 稀釋復性。上圖為一般稀釋。下圖為逆稀釋。 22
圖2-4-4微胞系統（reversed micelles system） 24
圖2-5 酵素法合成唾液酸之流程。 26
圖5-1 差向異構酶的表現之SDS-PAGE分析圖 58
圖5-2 溶解劑對差向異構酶包涵體溶解效應之SDS-PAGE分析圖 59
圖5-3 溶解劑對差向異構酶包涵體溶解效應 60
圖5-4 pH值對包涵體溶解之影響 61
圖5-5 SDS濃度對包涵體溶解之影響 62
圖5-6 最適包涵體溶解度 63
圖5-7 pH與差向異構酶濃度對復性之影響 64
圖5-8 不同pH值下復性之TBA呈色活性分析圖 65
圖5-9 再摺疊差向異構酶與天然差向異構酶 66
之大小排除層析圖譜 66
圖5-10 復性溫度對差向異構酶復性之可溶回收率影響 67
圖5-11 復性溫度對差向異構酶復性之活性影響 68
圖5-12 NaCl對差向異構酶復性之可溶回收率影響 69
圖5-13 NaCl對差向異構酶復性之活性影響 70
圖5-14 添加L-Arginine對差向異構酶復性之回收率影響 71
圖5-15 添加L-Arginine對差向異構酶復性之活性影響 72
圖5-16 添加PEG 1540對差向異構酶復性之回收率影響 73
圖5-17 添加PEG 1540對差向異構酶復性之活性影響 74
圖5-18 GSH與GSSG對差向異構酶復性回收率之影響 75
圖5-19 GSH與GSSG對差向異構酶復性活性之影響 76
圖5-20 GSH與GSSG對差向異構酶復性回收率之影響 77
圖5-21 GSH與GSSG對差向異構酶階段復性活性之影響 78
圖5-22 Epimerase 活性分析之HPLC層析圖譜 79
圖5-23 添加劑對復性的影響 80
圖5-24 GSH與GSSG對差向異構酶階段復性活性之影響 81
圖5-25 氧化復性之size exclsion chromotagraphy 層析圖譜 82
圖5-26a native epimerase螢光光譜 83
圖5-26b native epimerase螢光光譜 83
圖5-27 Dimer factions 之相對比活性 85
表目錄
表1 使用之添加劑和建議濃度…………………………………………… 18
表2 小分子添加劑的種類………………………………………………… 19
表3 HPLC分析條件………………………………………………………… 41
表4 大小排除管柱層析沖提條件………………………………………… 41
表5 以PEG做添加劑之文獻比較 ……………………………………………51

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