[[alternative]]Cloning and polymorphism of the ALD5 gene and heterologous expression, purification, and catalytic characterization of ALD5p of saccharomyces cerevisiae.

碩士[[abstract]]ALDH家族中包含了五個家族成員：（一)ALD1、（二）ALD2、（三）ALD3、（四）ALD4、（五）ALD5。本實驗之目的在於探討ALD5酵素對betanine aldehyde和3-aminopropanal之活性。根據之前研究報導,betanine aldehyde經由BADH的活化形成glycine-betanine,3-aminopropanal在ALD2和ALD3的作用下會形成β-alanine；然而並無人將ALD5作用於betanine aldehyde和3-aminopropanal。ALD5為位於第五號染色體位置上的基因，主要表現在粒線體組織內，其主要作用在於呼吸鏈的官能基生成、及在無氧環境下醋酸生成有關。 本實驗分成選殖和蛋白質表現兩部分進行： 一、在選殖部分：根據SGD基因序列而設定一段引子，經由連鎖聚合酶反應選殖。意外的是，此基因經定序發現有單一核酸多型性(SNP)存在，之後利用不同菌株來做進一步的比對加以確認。 二、在蛋白質表現部分：本實驗室利用四種不同的蛋白表現宿主菌株： BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)、BL21-CodonPlus，來做異源表現，使用乳糖和IPTG兩種不同誘導方式去探討不同誘導方式所生的蛋白質表現量，以及對其所誘導所表現出的ALD5蛋白來做活性測試，根據酵素動力學求出其Vmax和Km值。[[abstract]]The ALDH family including five family members: （1)ALD1,（2）ALD2,（3）ALD3,（4）ALD4,（5）ALD5. The aim of this study is to see whether there are any activities of ALD5p to betanine aldehyde and 3-aminopropanal. According to the past research , betanine aldehyde can be converted to glycine-betanine by BADH, and 3-aminopropanal converted to β-alanine by ALD2p and ALD3p. However, no one have determined if ALD5p is active towards betanine aldehyde and 3-aminopropanal. The ALD5 gene is located on chromosome V, and is localized to mitochondria. The ALD5p plays a regulatory role in mitochondrial respiration and is associated with the production of acetate on anaerobic conditions. This study contains two parts: 〈One〉. Cloning : Primers were designed according to ALD5 sequence on SGD for PCR cloning. Surprisingly, we found the evidence of existence of SNP (single nucleotide polymorphism). Then, SNP was confirmed using a different strain. 〈Two〉. Protein expression and purification: Our lab uses four strains as the host（BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)、BL21(DE3)、BL21-CodonPlus for heterlogous expression, and try to produce the protein by using different inducers, such as lactose and IPTG. Besides, we also use the purifed ALD5p to do dehydrogenase activity assay and find out the Vmax and Km on several substrates.[[tableofcontents]]中文摘要………………………………………………………iv 英文摘要………………………………………………………v 第一章 序論……………………………………………………………1 儀器……………………………………………………………7 藥品……………………………………………………………9 藥品配置………………………………………………………10 第二章 基因轉殖 實驗一、基因連鎖聚合酶反應 (PCR)………………………13 實驗二、PCR產物純化………………………………………14 實驗三、PCR產物限制酶酵素切割…………………………14 實驗四、DNA 接合反應 (TA Cloning)………………………16 實驗五、勝任細胞製備………………………………………16 實驗六、轉型作用 (Transformation)………………………20 實驗七、藍白篩選……………………………………………21 實驗八、質體小量製備………………………………………22 實驗九、表現載體的建構……………………………………24 實驗十、表現載體之確認 (1)PCR 分析法 (2)限制酶切割分析 結果與討論………………………………………………………26 第三章 重組蛋白表現 建構重組蛋白……………………………………………………39 實驗十一、生長曲線測定………………………………………39 實驗十二、SDS-PAGE……………………………………………40 實驗十三、酵素活性測定………………………………………44 實驗十四、蛋白質純化…………………………………………45 實驗十五、酵素動力學…………………………………………47 結果與討論………………………………………………………51 綜合討論…………………………………………………………72 參考文獻…………………………………………………………74[[note]]學號: 692290256, 學年度: 9