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研究生:陳怡君
研究生(外文):Yi-Chun Chen
論文名稱:聚乙烯醇固定化金屬親和吸附材於蛋白質純化之應用
論文名稱(外文):Polyvinyl alcohol-based immobilized metal affinity adsorbents for protein purification
指導教授:林松池
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:83
中文關鍵詞:固定化金屬親和層析法聚乙烯醇
外文關鍵詞:Immobilized Metal Ion Affinity ChromatographyPolyvinyl alcohol
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本研究利用聚乙烯醇 (polyvinyl alcohol,PVA)在酸性下與戊二醛 (glutaraldehyde,GA) 進行化學修改,產生類似交聯 (crosslinking) 以及接枝式 (graft) 結構的水膠 (hydrogel) 型高分子粉末作為固態載體。利用化學合成的方式接上亞胺基二乙酸 (IDA) 螯合劑,作為固定化金屬親和吸附材,螯合銅離子並應用於純化Racemase與 Epimerase的粗酵素液。文中探討各種影響蛋白質吸附量與吸附因素,例如:交聯比例、金屬離子、pH值、氯化鈉濃度等。經實驗結果,獲得最適化吸附基材交聯比例為5 %聚乙烯醇、13 mM戊二醛,交聯4小時後含水率約為78 %。螯合銅離子最適化吸附與清洗條件為37 ℃、0.05 M Tris-HCl、300 mM NaCl、pH 8,脫附條件為25 ℃、0.05 M Tris-HCl、300 mM NaCl、300 mM imidazole、pH 8,總蛋白質脫附量為52 μg / g-cPVA gel。經上述結果,cPVA gel作為固定化金屬親和吸附材應用於基因重組聚組織胺標的蛋白質的吸附,多數被吸附於基材上,可選擇不同孔洞性質的膠體,降低吸附基材上的非特定蛋白質吸附,以達到純化蛋白質之目的。
In this study, crosslinking structure or graft structure beads were prepared by a crosslink polymerization technique and modified by IDA (iminodiacetic acid), loading with Cu2+, and used as adsorbent to purify a target protein (Racemase and Epimerase) from the large volumes of liquid culture medium. In the text, the such are factors that influence the adsorption of target proteins are discussed, for example: crosslinking ratio, category of metal ion, pH ionic, salt composition and NaCl concentration in the buffer. The results indicate that the optimum adsorbent crosslinking ratio is 5 % PVA and 13 mM GA crosslinked 4 hour, and water content is 78 %. The optimum purification procedure condition is 37 ℃、300 mM NaCl、pH 8、0.05 M Tris-HCl buffer for loading, and 25 ℃、300 mM NaCl、300 mM imidazole、pH 8、0.05 M Tris-HCl buffer for elution, which results in a total protein elution for 52 μg / g-cPVA gel.
目錄
中文摘要...............................................Ⅰ
Abstract...............................................Ⅱ
目錄...................................................Ⅲ
表目錄.................................................Ⅷ
圖目錄.................................................Ⅷ
第一章 諸論............................................1
第二章 文獻回顧與原理..................................3
2-1 蛋白質純化.......................................3
2-1-1蛋白質簡介..................................3
2-1-2蛋白質純化技術..............................5
2-2 固定化金屬親和層析法
(Immobilized Metal Ion Affinity Chromatography)........10
2-2-1固定化金屬親和層析法簡介....................10
2-2-2固體相膠體的組成............................11
2-2-3吸附基材....................................11
2-2-4延伸臂......................................12
2-2-5金屬螯合劑..................................13
2-2-6過渡金屬離子................................14
2-3 影響吸附的因素...................................15
2-3-1蛋白質結構...................................16
2-3-2 pH值........................................17
2-3-3緩衝溶液與鹽類...............................17
2-4 影響脫附的因素...................................18
2-5 研究系統簡介.....................................19
2-5-1聚乙烯醇 (Polyvinyl alcohol,PVA).............19
2-5-2戊醛 (Glutaraldehyde,GA) ....................20
2-6 標的蛋白之簡介...................................21
2-6-1 N - Acylamino acid racemase (Racemase)簡介...21
2-6-2 N - acetyl D- glucosamine 2- epimerase
(Epimerase)簡介..............................22
第三章 實驗藥品與儀器..................................24
3-1 實驗藥品.........................................24
3-2 實驗儀器.........................................25
第四章 實驗方法與分析..................................27
4-1 酵素液的製備.....................................27
4-1-1 LB固態洋菜培養基的製備.....................27
4-1-2 LB液態培養基的製備.........................27
4-1-3 Racemase菌種培養...........................27
4-1-4蛋白質的收集與純化..........................28
4-1-4.1 利用商用膠體進行酵素純化............29
4-2 cPVA膠體合成.....................................30
4-2-1 基材的合成.................................30
4-2-2 基材表面活化的合成.........................31
4-2-3 螯合劑的合成...............................31
4-2-4 金屬離子的鍵結.............................31
4-3 cPVA膠體之基本性質測試...........................32
4-3-1交聯劑濃度及反應時間對產率 (yield) 的測試...32
4-3-2表面積及孔洞性質測定........................32
4-3-3含水率 (water content) 測試.................33
4-3-4 銅離子鍵結量測定...........................33
4-4 金屬離子最適化...................................34
4-5 銅離子最適化吸附脫附條件.........................35
4-5-1 添加二氮二烯五圜 (imidazole) 濃度效應......35
4-5-2 pH值效應...................................37
4-5-3 鹽類效應...................................38
4-5-4 以乙二胺四乙酸 (EDTA) 脫附蛋白質...........39
4-5-5 以煮沸法脫附蛋白質.........................40
4-6 鈷離子離子最適化吸附脫附條件.....................41
4-6-1添加二氮二烯五圜濃度效應....................41
4-6-2鹽類效應....................................42
4-6-3 pH值效應...................................43
4-6-4 以十二烷基硫酸鈉 (SDS) 脫附蛋白質..........44
4-6-5 溫度效應...................................46
4-7 比較Cu-cPVA與Ni-NTA吸附效應......................47
4-8 延伸臂效應.......................................48
4-9 實驗分析.........................................48
4-9-1 蛋白質電泳分析 (SDS-polyacryamide gel
electrophresis)............................48
4-9-2 蛋白質定量分析.............................50
4-9-3 TINA影像分析...............................51
第五章 結果與討論......................................52
5-1 金屬親和吸附材之基本性質測定.....................52
5-1-1 交聯產率的量測.............................52
5-1-2 膠體表面積與表面結構量測...................55
5-1-3 膠體的含水率...............................56
5-1-4 銅離子鍵結量的量測.........................57
5-2 金屬離子最適化...................................58
5-3 銅離子之最適化吸附與脫附條件.....................60
5-3-1 添加二氮二烯五圜濃度效應...................60
5-3-2 pH值效應..................................61
5-3-3 鹽類效應...................................61
5-3-4以乙二胺四乙酸脫附蛋白質....................63
5-4 鈷離子之最適化吸附與脫附條件.....................64
5-4-1以二氮二烯五圜吸附與乙二胺四乙酸之脫附效應..64
5-4-2鹽類效應....................................66
5-4-3 pH值效應...................................68
5-5 以十二烷基硫酸鈉脫附蛋白質.......................69
5-6 銅離子與鈷離子之溫度效應.........................71
5-7 比較Cu-cPVA與Ni-NTA吸附效應......................73
5-8 延伸臂效應.......................................73
第六章 結論............................................74
第七章 參考文獻........................................77
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