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研究生:陳心怡
研究生(外文):Hsin-Yi Chen
論文名稱:胃幽門桿菌菌種26695之HP0291蛋白質結構似AroQ型chorismate變位酵素功能性鑑定與分析
論文名稱(外文):HP0291, a newly isolated, AroQ structure class-like chorismate mutase of Helicobacter pylori strain 26695
指導教授:呂平江
指導教授(外文):Ping-Chiang Lyu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:68
中文關鍵詞:胃幽門桿菌 蛋白質 酵素
外文關鍵詞:chorismateHelicobacter pylori
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胃幽門桿菌菌種26695,是一種微厭氧菌,分類上屬於革蘭氏陰性的螺旋狀桿菌。它是胃潰瘍的主要病因以及胃癌的一個早期病徴,現今是存在於人類中最普遍的一種由細菌傳染而造成的慢性疾病。自從1997年Tomb, J. F 發表了此菌的整個基因體序列後。我們根據基因序列,資料庫搜查,基因註解,序列預測和比對的方法,致力於胃幽門桿菌結構基因體的研究。在本文中,胃幽門桿菌基因體內我們所選的基因之一 (HP0291),己成功地確認了它的功能:胃幽門桿菌菌種26695的chorismate變位酵素。
HP0291蛋白質,為胃幽門桿菌菌種26695基因HP0291所轉譯出的一個功能未知蛋白質,現在已經成功的被選殖、純化及鑑定出此蛋白質的功能。HP0291蛋白質已被純化成在聚丙烯洗胺凝膠體上的單一蛋白質,並且推估其分子量約為11.3 kDa。每一升的Luria-Bertani培養基約可純化出純的HP0291蛋白質25毫克。圓二色光譜儀分析HP0291蛋白質的結果指出HP0291為一個典型α-螺旋的結構,其螺旋度大約為39百分比,並且它的半變性溫度大於攝氐溫度75度。分析型超高速離心機和化學聯結的實驗顯示HP0291蛋白質有多單體的現象,且主要形為雙體。化學法突變HP0291蛋白質硫基成羧基的HP0291蛋白質(HP0291-m)顯示出和HP0291蛋白質相似的蛋白質二級結構和對熱的穩定性,當反應緩衝溶液中不含effectors時,HP0291-m較HP0291蛋白質有更佳的酵素活性。酵素動力學實驗分析得到HP0291蛋白質和HP0291-m蛋白質的Michaelis-Menten常數分別是355 µM; 284 µM,Kcat/Km值分別為30.24 sec-1 mM-1; 39.61 sec-1 mM-1。chorismate變位酵素的演化樹分析結果顯示出HP0291蛋白質在結構上似AroQ-類型的chorismate變位酵素。
Helicobacter pylori, a microaerophilic, Gram-negative and spiral-shaped organism. It is the major cause of gastric ulcers and an early risk factor for gastric cancer. Since the whole genome sequences of H. pylori strain 26695 have been reported by Tomb, J. F in 1997. We aim on structural genomics study of H. pylori strain 26695 based on database search, gene annotation, sequence prediction and alignments. In this thesis, one of our targets (HP0291) had been successfully cloned, characterized and confirmed its function: The chorismate mutase in H. pylori.
HP0291, encoding hypothetical protein from H. pylori strain 26695, was purified to homogeneity on a SDS polyacrylamide gel with a deduced molecular mass of 11.3 kDa and yield about 2 mg native protein per liter Luria-Bertani Medium. Circular dichroism spectra indicate HP0291 is a typical α-helix structure protein with the helicity about 40 %, and the melting temperature is higher than 75°C. Analytical ultracentrifuge and cross-linking experiments on the purified HP0291 protein reveal that it has oligomer phenomenon, and the major form is dimer. By cysteine modification, the S-Carboxymethylated HP0291 (HP0291-m) shows similar secondary structures and thermal stabilities to HP0291 but contain better enzyme activities compare to HP0291 especially when the reaction buffer containing no effectors. The kinetic data show Michaelis-Menten constant (Km) values of HP0291 and HP0291-m are 355 µM; 284 µM, and a Kcat/Km values of 30.24 sec-1 mM-1; 39.61 sec-1 mM-1 respectively. The phylogenetic tree of chorismate mutases characterized so far indicated that HP0291 shows similarities to the AroQ-type enzyme.
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