酵母菌ALD5基因選殖許多型性、異源表現、純化與酵素活性之探討

Cloning and polymorphism of the ALD5 gene and heterologous expression, purification, and catalytic characterization of Ald5p of Saccharomyces cerevisiae.

何瑞文(Jui-Wen Ho)

指導教授 ： 陳銘凱

淡江大學/理學院/生命科學研究所碩士班/碩士(2005年)

https://doi.org/10.6846/TKU.2005.00302

摘要

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。

關鍵字

選殖 ； 純化 ； 酵素動力學

並列摘要

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.

並列關鍵字

Aldehyde dehydrogenase ； Yeast Ald5p ； ALD5 gene ； Single nulotide polymorphism

參考文獻

Kurita O and Nishida Y (1999)Involvement of mitochondrial aldehyde dehydrogenase ALD5 in maintenance of the mitochondrial electron transport chain in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 181, 281–287.

Tessier, W.D., Meaden, P.G., Dickinson, F.M. and Midgly, M. (1998) Identi¢cation and disruption of the gene encoding the K.-activated acetaldehyde dehydrogenase of Saccharomycescerevisiae. FEMS Microbiol. Lett. 164, 29-34.

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Kurita, O. and Ito, H. (1994) Isolation and characterization of mutants partially de¢cient in aldehyde dehydrogenase in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 58, 609-615.

Tessier, W. D., P. G. Meaden, F. M. Dickinson and M. Midgley, 1998 Identification and disruption of the gene encoding the K-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae.