淡江大學機構典藏:Item 987654321/32589
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    题名: 啤酒酵母菌醇類去氫酶3 與4之選殖、過度表現、純化及動力學分析
    其它题名: Cloning, overexpression, purification and kinetic analysis of alcohol dehydrogenase 3 and 4 from Saccharomyces cerevisiae
    作者: 邱明諄;Chiu, Ming-chun
    贡献者: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    关键词: 啤酒酵母菌;醇類去氫酶;Saccharomyces cerevisiae;Alcohol dehydrogenase
    日期: 2005
    上传时间: 2010-01-11 02:24:08 (UTC+8)
    摘要: 本研究主要目的在同源表現啤酒酵母菌醇類脫氫酶3 與4（Alcohol Dehydrogenase3、4），並測定其對膽鹼（Choline）的活性。以聚合酶連鎖反應（PCR）進行酵母菌菌株YPH499 的ADH3 和ADH4 基因放大，利用pGEM-T easy vector 做TA-cloning，再將質體DNA 定序，其結果與資料庫SGD 進行比對，以確認序列的正確性。比對結果：所選殖之ADH3 基因與SGD 符合，而ADH4 則有單一核酸多型性（Single Nucleotide Polymorphism, SNP）的現象。接著將ADH3 和ADH4 重組於pYES2 表現載體，再以2％galactose 誘導其在酵母菌（BJ2168）中大量表現。目標蛋白質位於粒線體胞器中[2]，因此在純化策略上，先溶除細胞壁形成原生質球狀體，以便分離粒線體，再採取以超音波粉碎機處理，分離出粒線體粗抽蛋白後，以蛋白質液體色層分析儀（Fast Protein Liquid Chromatography, FPLC）純化之，再檢測其對受質膽鹼的動力學活性。並討論此活性與甜菜鹼（Betaine）代謝之關係。
    The main purpose of this research is homologous expression Saccharomyces cerevisiae alcohol dehydrogenase 3 and 4, and determine its activity to choline. Using polymerase chain reaction（PCR）to amplify ADH3 and ADH4 gene from yeast strain YPH499, we utilized pGEM-T easy vector for TA-cloning and sequenced the inserted DNA. The results of sequencing were compared with database SGD in order to confirm the exactness of PCR. We found ADH3 gene that we cloned conforms to databank SGD,but ADH4 has a Single Nucleotide Polymorphism（SNP）. ADH3 and ADH4 were subcloned to expression vector pYES2 , and then induce them to over-express in the Saccharomyces cerevisiae（BJ2168）with 2%galactose. Since both enzymes are mitochondrial proteins, we firstly isolated mitochondria by preparation of spheroplast,then broke the mitochondria by sonication. The protein was purified by Fast Protein Liquid Chromatography（FPLC）, and the enzyme activities determined for ethanol and choline.The kinetic properties are discussed in relation to choline-betaine pathway.
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