啤酒酵母菌醛類去氫酶2和3之選殖，過度表現，純化及酵素活性分析

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題名:	啤酒酵母菌醛類去氫酶2和3之選殖，過度表現，純化及酵素活性分析
其他題名:	Cloning, overexpression, purification and catalytic analysis of aldehyde dehydrogenase 2 and 3 from saccharomyces cerevisiae
作者:	毛清源;Mao, Ching-yuan
貢獻者:	淡江大學生命科學研究所碩士班陳銘凱;Chern, Ming-kai
關鍵詞:	啤酒酵母菌;乙醛去氫酶2;乙醛去氫酶3;acetaldehyde dehydrogenase;ALD2;ALD3
日期:	2008
上傳時間:	2010-01-11 02:28:31 (UTC+8)
摘要:	啤酒酵母菌醛類去氫酶2（YMR170C）和醛類去氫酶3（YMR169C）皆為ACDH ( acetaldehyde dehydrogenase )家族中的一員，並被認為用NADP+當作輔酶。最近的文獻也顯示ALD2和ALD3會使泛酸的生合成途徑中的3-aminopropanal 轉變成β-alanine。首先使用PCR將基因片段大量複製， ALD2和ALD3的基因片段長度皆為1521個鹼基，再用TA-cloning將其接於pGEM-T Easy Vector上，成為pGEM-T-ALD2和pGEM-T-ALD3。避免影響醛類去氫酶的蛋白質結構，所以必須在最不破壞其基因序列的狀況下表現，將表現載體pYES2和pGEM-T-ALD2利用限制酶SacI、EcoRI進行切割，再用T4 DNA ligase接合成為質體pYES2-ALD2和pYES2-ALD3。將質體pYES2-ALD2和pYES2-ALD3先送入酵母菌宿主（BJ2168 strain），以2％galactose 誘導讓其在酵母菌中表現，並在大量表現之後，但是測不到活性。於是利用SacI、XbaI將質體pYES2-ALD3作切割後，利用T4 DNA ligase接合成為pYeSL-ALD3並與pYES2-ALD2同時送入酵母菌宿主strain（BJ2168）使得兩蛋白質同時在同一個酵母菌作大量表現。同樣地，我們也建構pYeSL-ALD2並與pYES2-ALD3在同一個酵母菌作大量表現。接著我們測定共表現之產物活性，並以質譜測定是否為正確的共表現組成。 In Saccharomyces cerevisiae ALD2 (YMR170C) and ALD3 (YMR169C) are the members of ACDH ( acetaldehyde dehydrogenase ) family, and the gene products supposed to use NADP+ as coenzyme. Recent literature also shows that ALD2 and ALD3 genes are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to β-alanine in vivo. In this research, we cloned the ALD2 and ALD3 gene by polymerase chain recitation (PCR). The size of both DNA fragments are 1,521bp. The ORFs of ALD2 and ALD3 gene were cloned into pGEM-T easy vector by TA-cloning to obtained pGEM-T-ALD2 and pGEM-T-ALD3 plasmids In order to express protein in yeast, we constructed plasmid pYES2-ALD2 and pYES2-ALD3 using SacI、EcoRI restriction sites from pGEM-T-ALD2 and pGEM-T-ALD3. We made use of 2% galactose to induce Ald2p and Ald3p to over-express in the S. cerevisiae （BJ2168 strain）. But no catalytic activity can be found in both Ald2p and Ald3p. So we constructed plasmid pYeSL-ALD3 by SacI、XbaI restriction enzymes from pYES2-ALD3. We used 2% galactose to induce Ald2p (pYES2-ALD2) and Ald3p (pYeSL-ALD3) to co-over-express in BJ2168. Similarly, we constructed plasmid pYeSL-ALD2 and co-over-express with Ald3p (pYES2-ALD3). We then tested the activity of the co-over-expressed product and deterimined the components by mass spectrometry.
顯示於類別:	[生命科學研究所] 學位論文

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