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研究生:陳麗安
研究生(外文):Li-An Chen
論文名稱:鈣、鎂離子對回收間質蛋白質的影響
指導教授:陳特良
指導教授(外文):Teh-Liang Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
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:2002
畢業學年度:90
語文別:中文
論文頁數:40
中文關鍵詞:鈣離子滲透壓衝擊間質蛋白質鎂離子大腸桿菌
外文關鍵詞:periplasmic proteinsosmotic shockCa+2 ionE.coliMg+2 ion
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在以大腸桿菌為宿主細胞生產基因蛋白質並將其回收的系統中,一般回收方式會將基因蛋白質和胞內蛋白質一起取出來,使得基因蛋白質的純度過低,處理困難。本研究是以滲透壓衝擊(osmotic shock)並同時添加EDTA的方式,使E. coli JM109中細胞間質的肌酸酵素釋放至菌體外,另外並以添加鈣、鎂離子作前處理和在液態培養基中添加鈣、鎂離子兩種方式,來增加肌酸酵素的回收與純度。吾人發現分別添加鈣、鎂離子作前處理時能分別提高肌酸酵素63%、43%的回收率,而純度也分別提高30%及 27%。在液態培養基中添加鈣、鎂離子亦可增加肌酸酵素回收率及純度,但細胞生長在鈣、鎂離子濃度下時,會使肌酸酵素活性降低,因此以添加鈣、鎂離子作前處理的方式是較在液態培養基中添加時來得好。另外同時添加鈣、鎂離子作前處理並不能增加肌酸酵素的回收,其原因為鈣、鎂離子對於細胞外膜的吸附具有競爭力。因此僅以添加鈣離子作前處理時能得到較高回收率及純度的肌酸酵素。
Numerous foreign proteins are secreted to periplasmic space of recombinant Escherichia coli. This work was to extract a periplasmic enzyme, creatinase, without releasing cytoplasmic proteins. We adapted osmotic shock that releases protein solely by changing environment osmotic pressure. Addition of EDTA helps to reducing diffusion resistant by chelating divalent cation out off the outer membrane. To improve the enzyme recovery, we tried to increase the divalent cation concentration of outer-membrane surface. One approach was to pretreat cells with Ca+2, Mg+2 solution. The enzyme recovery was increased by 63% and 43%, respectively; purity could be increased by 30% and 27%. Another approach was to incubate the cells in medium abundant in Ca+2 or Mg+2. Although it also increased the enzyme recovery and purity, the high concentration of cation used decreased product yield. Comparing the effects of Ca+2 and Mg+2, the former had a better recovery. Using Ca+2 and Mg+2 simultaneously, the enzyme recovery could not be improved; this is due to the absorptions of Ca+2 and Mg+2 to outer membrane are competitive. The existence of Mg+2 decreases the surface concentration of Ca+2, there fore; it’s better to use Ca+2 as the only cation to pretreat the cell.
中文摘要 I
Abstract II
表目錄 IV
圖目錄 V
第一章 緒論 1
1-1 肌酸酵素 1
1-2 重組基因蛋白 1
1-3 細胞壁的結構及作用 2
1-3-1 Peptidoglycan 2
1-3-2 細胞外膜(lipopolysaccharide layer) 3
1-3-3 細胞間質和蛋白質的釋放 4
1-4 酵素的回收 4
1-5 研究背景及動機 6
第二章 材料與方法 10
2-1 宿主載體系統: 10
2-2 藥品 10
2-3 實驗儀器與裝置 11
2-4 實驗步驟 12
2-4-1 菌種的培養與保存 12
2-4-2 細胞處理方法 12
2-5-1 菌體濃度檢量線 13
2-5-2肌酸酵素的活性分析 13
2-5-3蛋白質的分析 14
第三章 結果與討論 18
3-1 不同培養時間下的細胞對osmotic shock敏感度影響 18
3-2 EDTA處理時間對活性釋放的影響 18
3-3 以鈣離子做前處理 19
3-4 EDTA的量對osmotic shock的影響 19
3-5 前處理時間對酵素釋放的影響 20
3-6 LB中添加鈣離子的影響 20
3-8 不同金屬離子的影響 21
3-9 混合兩種金屬離子的影響 22
第四章 結論 36
參考資料 37
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