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系統識別號 U0002-1508201212423700
中文論文名稱 逆沖提法於吸附型管柱層析蛋白質純化之探討
英文論文名稱 The Study of Protein Purification in Adsorption Column Chromatography by Back Flush
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 100
學期 2
出版年 101
研究生中文姓名 張慧蘭
研究生英文姓名 Hui-Lan Chang
學號 699160130
學位類別 碩士
語文別 中文
口試日期 2012-06-20
論文頁數 92頁
口試委員 指導教授-陳銘凱
委員-林賜恩
委員-官宜靜
委員-陳銘凱
中文關鍵字 純化  逆沖提法  吸附性管柱 
英文關鍵字 purifie  Back Flush  Adsorption Column  Chromatography 
學科別分類 學科別自然科學化學
中文摘要 利用帶有YNR029Cp、ALD4p基因的質體轉形至大腸桿菌(E.coli)的表現宿主細胞BL21(DE3)表現並純化後,以Q SepharoseTM Fast Flow、Hydroxylapatite(使用純化之YNR029Cp)、Phenyl Sepharose 6 Fast Flow、Blue Sepharose 6 Fast Flow(使用過度表現之ALD4p)進行分離,以反向吸附純化的蛋白質樣品,正向沖提蛋白質。配合不同pH值和流速,找出並測試是否對樣品流出的前後順序有關及其分離的條件。
英文摘要 Plasmids carrying YNR029Cp or ALD4p gene were transformed into E.coli BL21(DE3), and the individual protein was then expressed and purified. Q-Sepharose Fast Flow, Hydroxylapatite (for purified YNR029Cp), Phenyl Sepharose 6 Fast Flow, Blue Sepharose 6 Fast Flow (for over-expressed ALD4p) were employed in order to reversely adsorb the purified protein sample, followed by forward elution of the protein. With the different pH and flow rate, we tried to identify and test the relationship to the order of elution and the separation conditions.
論文目次 謝誌 II
中文摘要.......................................................................................................VI
英文摘要........................................................................................................V
目錄 VI
圖目錄............................................................................................................IX
縮 寫 表 X
第一章 前言 1
1.1 研究目的 1
1.2 乙醛去氫酶ALD分為兩大類 1
1.2.1 來自粒線體 1
1.2.2 來自細胞質 1
1.3 YNR029c 2
1.4 YHR209W 2
1.5 YIL064W 2
1.6 大腸桿菌 3
1.7 酵母菌 4
第二章 材料和方法 5
2.1 製備YNR029Cp 5
2.1.1 誘導YNR029Cp的表現 5
分析粗抽蛋白質 6
2.1.2 超音波破菌法 6
2.1.3 His-tag純化 6
2.1.4 SDS蛋白質電泳 7
2.1.5 透析 8
2.1.6 測蛋白質濃度 10
2.2 製備YHR209Wp(Rosetta) 11
2.2.1 誘導YHR209Wp的表現 11
2.2.2 超音波破菌法 12
2.2.3 His-tag純化 12
2.2.4 SDS蛋白質電泳 13
2.2.5 透析 13
2.2.6 測蛋白質濃度 13
2.3 製備SEE1p 13
2.3.1 誘導SEE1p的表現 13
2.3.2 超音波破菌法 14
2.3.3 His-tag純化 14
2.3.4 SDS蛋白質電泳 14
2.3.5 透析 14
2.3.6 測蛋白質濃度 15
2.4製備ALD4p 15
2.4.1誘導目標蛋白質ALD4p表現 15
分析粗抽蛋白質 16
2.4.2 超音波破菌法 16
2.5蛋白質純化 16
2.5.1離子交換樹脂 16
2.5.1.1 配製Q SepharoseTM Fast Flow所需buffer 16
2.5.1.2 陰離子交換樹脂純化 16
管柱裝填 17
2.5.1.3 進行蛋白質SDS-PAGE分析 20
2.5.1.4銀染 20
2.5.2 Hydroxylapatite 21
2.5.2.1 配製Hydroxylapatite所需buffer 21
2.5.2.2 Hydroxylapatite 樹脂純化 21
2.5.2.3跑蛋白質電泳 23
2.5.2.4銀染 23
2.5.3 Phenyl Sepharose 6 Fast Flow 23
2.5.3.1 配製Phenyl Sepharose 6 Fast Flow所需buffer 23
2.5.3.2 Phenyl Sepharose 6 Fast Flow純化 24
2.5.3.3跑蛋白質電泳 24
2.5.3.4銀染 24
2.5.4 Blue Sepharose 6 Fast Flow 24
2.5.4.1 配製Blue Sepharose 6 Fast Flow所需buffer 24
2.5.4.2 Blue Sepharose 6 Fast Flow純化 25
2.5.4.3跑蛋白質電泳 25
2.5.4.4銀染 25
2.6 應用Image J定量蛋白質電泳條帶 26
第三章 實驗之結果分析 27
3.1 誘導目標蛋白質YNR029Cp表現 27
3.2 YNR029Cp用His-tag純化 27
3.3 誘導目標蛋白質ALD4p表現 27
3.4 Q SepharoseTM Fast Flow、Hydroxylapatite(使用YNR029Cp)、Phenyl Sepharose 6 Fast Flow、Blue Sepharose 6 Fast Flow(使用ALD4p)純化之比較 27
第四章 其他因素的討論 35
第五章 結論 37
第六章 未來展望 39
第七章 參考文獻 39
第八章 圖 44

圖目錄:
圖 1. YNR029c 用His-tag 純化 ............................................................................................. 44
圖 2. 誘導ALD4 表現 ........................................................................................................... 45
圖 3. 純化之YNR029cp 進行陰離子交換樹脂-體積比 ..................................................... 46
圖 4. 不同樣品體積之陰離子交換比較(0.05 M→1 M NaCl) ....................................... 52
圖 5. ALD4p 進行Phenyl Sepharose 6 Fast Flow-體積比 ................................................. 53
圖 6. 3.5 mL 之Phenyl Sepharose Fast Flow 比較圖(0.2 M→0 M K3PO4) ................ 59
圖 7. 10 mL 之Phenyl Sepharose Fast Flow 比較圖(0.2 M→0 M K3PO4) ................. 60
圖 8. Phenyl Sepharose Fast Flow 比較圖(0.2 M→0 M K3PO4)................................... 61
圖 9. ALD4p 進行Blue Sepharose 6 Fast Flow-體積比 ..................................................... 62
圖 10. Blue Sepharose Fast Flow 比較圖(0 M→1.5 M NaCl) ....................................... 65
圖 11. 純化之YHR209Wp+ALD4p 進行陰離子交換樹脂-電荷比 .................................. 66
圖 12. 純化之SEE1p+YNR029cp 進行陰離子交換樹脂-電荷比 ..................................... 67
圖 13. 不同蛋白質之比較圖 ................................................................................................. 68
圖 14. 純化之YNR029cp 進行Hydroxylapatite-孔隙率比............................................... 69
圖 15. Hydroxylapatite 比較圖 .............................................................................................. 73
圖 16. 3.5 mL ,Hydroxylapatite 與陰離子之比較圖 ............................................................ 74
圖 17. 10 mL ,Hydroxylapatite 與陰離子之比較圖 ............................................................. 75
圖 18. 純化之YNR029cp 進行陰離子交換樹脂-流速比 ................................................... 76
圖 19. 不同流速之陰離子交換之比較圖 ............................................................................. 80
圖 20. 純化之YNR029cp 進行陰離子交換樹脂-管柱粗細 ............................................... 81
圖 21. 不同粗細管柱之陰離子交換比較圖 ......................................................................... 82
圖 22. 純化之YNR029cp 進行陰離子交換樹脂-停留時間 ............................................... 83
圖 23. 不同停留時間之陰離子交換比較圖 ......................................................................... 84
圖 24. 純化之YNR029cp 進行陰離子交換樹脂-pH 值 ..................................................... 85
圖 25. 不同pH 值之陰離子交換比較圖 .............................................................................. 87
圖 26. ALD4p 進行Phenyl-鹽濃度 ....................................................................................... 88
圖 27. 不同鹽濃度之Phenyl Sepharose Fast Flow 比較圖 ............................................... 92
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