臺灣博碩士論文加值系統

Gene I蛋白共348個氨基酸，位於E. coli的內膜上，與噬菌體f1感染E. coli後移出宿主時，組裝病毒粒(particle)的過程有關。E. coli 的Thioredoxin會影響f1組裝子代，可能與f1的Gene I蛋白有交互作用。有研究指出，Gene I 與許多ATPase或ATP-binding protein在蛋白質N-端有著相似的氨基酸序列，所以我們懷疑Gene I具有ATPase的活性。
由於Gene I wildtype蛋白只要大量表現，宿主細胞便會停止生長甚至死亡，無法大量表現及純化出Gene I蛋白，但我們已順利地表現出線形噬菌體f1 Gene I 兩個突變株的蛋白，分別是表現出125及255個胺基酸的Gene I蛋白(稱作Gene I126及Gene I256)。實驗結果發現可分解ATP，證實Gene I確實有ATPase的活性，且Gene I256之ATPase活性較Gene I126高，可能是因為Gene I256比較接近完整wildtype蛋白。另外加入不同濃度的thioredoxin會增加Gene I之ATPase 活性。

中文摘要
英文摘要
致謝
目錄
表目錄
圖目錄
縮寫表
一、緒論
1.1 線形噬菌體f1········1
1.2 線形噬菌體f1之Gene I蛋白·······2
1.3 Thioredoxin簡介·······3
1.4 大腸桿菌Thioredoxin·······4
1.5 大腸桿菌Thioredoxin和線形噬菌體f1之關係·······5
1.6 大腸桿菌Thioredoxin和Gene I蛋白之關係···6
二、研究目的·······7
三、材料與方法
3.1 材料
1.菌株······8
2.質體·······9
3.培養基·······12
4.溶液與緩衝液·······12
5.Thioredoxin蛋白質緩衝液·······14
6.蛋白質電泳溶液······14
7.分析材料與酵素·······16
8.Glutathione Sepharose 4B蛋白質純化······16
9.6X His-Tag affinity column蛋白質純化·······17
10.ATP水解後磷酸根(Pi)產生之測試······17
11.試劑組(kits) ········18
12.ATPase活性分析藥品·····18
3.2 實驗方法
1.質體抽取······19
2.勝任細胞(competent cell)的製備········19
3.轉形作用·····20
4.載體的製備······20
5.DNA extraction kit(購自Viogene)--DNA回收·······20
6.f1噬菌體單股DNA(ssDNA)抽取·····21
7.f1噬菌體雙股DNA(RF DNA)抽取·····21
8.菌體培養······21
9.Gene I之表現·······23
10.Thioredoxin蛋白質粗抽液之製備及純化······24
11.蛋白質電泳······26
12.pGEX-5X-1/Gene I蛋白之純化······27
13.Glutathione Sepharose 4B的再生與保存······27
14.pET-30a/Gene I蛋白之純化·····28
15.His•Bind resin的再生(regeneration)·····29
16.還原氧化態Thioredoxin及去除樣品中DTT········29
17.ATPase活性之分析······29
18.ATP水解後磷酸根(Pi)產生之測試······30
四、實驗結果
1.製備pGEX-5X-1/Gene I126及Gene I256·····31
2.表現及純化BL21(DE3)-pGEX-5X-1/Gene I126及Gene I256···31
3.表現及純化Origami(DE3)-pGEX-5X-1/Gene I126及Gene I256·32
4.製備pET-30a/Gene I126及Gene I256····33
5.表現及純化BL21(DE3)-pET-30a/Gene I126及Gene I256···33
6.ATPase活性分析·······34
7.Thioredoxin對Gene I蛋白ATPase活性之影響······36
8.ATP水解釋出磷酸根之呈色反應··········36
9.Gene I126及Gene I256序列比對及結構模擬······37
五、討論··················39
六、結論················43
七、未來展望·········44
參考文獻·············45

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