酵母菌ALD6基因選殖與多型性、異源表現、純化與酵素特性之探討

Cloning and polymorphism of the ALD6 gene and heterologous expression,purification, and catalytic characterization of Ald6p of Saccharomyces cerevisiae.

陳煜群(Yu-Chun Chen)

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.2005.00381

摘要

自1950 年代，啤酒酵母菌 Aldehyde dehydrogenase (ALDH) 研究至今，已知是種pyruvate dehydrogenase (PDH) bypass 的關鍵酵素。至今，已發現5 個ALDH 成員( ALD 基因)。在細胞質中ALDH 所扮演的角色是在於產生乙醯輔酶 A。根據文獻報告指出此ALD6 基因位在第16 號染色體的 left arm 上， ALD6 基因所轉錄之 Ald6p 即為細胞質的 ALDH。
在本研究中，利用聚合酶連鎖反應 (PCR) 放大欲選殖之ALD6 基因片段。此 DNA 片段大小為 1,506 bp，包含在5＇端設計一個限制酶（ NdeI）作用反應序列。接著將 ALD6 基因接於pGEM®-T easy vector ，此法稱做TA-Cloning。經質體轉型至 E.coli DH5α取得大量含pGEM-T-ALD6 基因之質體。此外，在pGEM-T-ALD6 質體上含此兩種限制酶（ NdeI、SpeI）作用反應序列，透過限制酶反應作用後自pGEM-T-ALD6 質體上得到含有 NdeI 與SpeI 之 ALD6 基因片段。同樣在表現载體 pET-43.1c(+) 也含有此兩種限制酶（ NdeI、SpeI）作用反應序列，經過NdeI 與SpeI 作用後，可以得到此兩種限制酶所切下的專一性片段载體DNA。隨後透過接合酶T4 ligase 接合完成表現質體的pET-43-ALD6 的建構。接著再次轉型至 E.coli DH5α取得大量之表現質體 pET-43-ALD6，隨後再轉型送入四種E.coli 表現宿主菌株（BL21(DE3)、BL21pLysS、Rosetta(DE3)、BL21-CodonPlus(DE3)-RIL）以達到大量表現 Ald6p 重組蛋白之目的。在這四種含pET-43-ALD6 質體之表現菌株利用不同的誘發物質、培養溫度、最適培養時間以及初步酵素活性測試結果顯示，以30℃、Lactose 誘導Rosetta(DE3) 最佳，最適條件為誘導後的第12 小時，Ald6p 的反應速率與蛋白質含量皆比其他三種表現菌種來的好。
在已找尋最適表現 Ald6p 重組蛋白之條件後，利用該條件擴大製備 Ald6p。接著進行Ald6p 重組蛋白的純化，本研究使用 Amersham Biosciences 公司純化樹脂：1. Q Sepharose
fast flow 2. Blue Sepharose 6 fast flow。已純化的 Ald6p 利用不同的受質之醛類（Propionaldehyde、4-aminobutyraldehyde、3-aminopropionaldehdye、Betaine aldehyde 等）進行一系列酵素動力分析，求得 Km 與 Vmax 值，藉以了解不同受質之醛類對Ald6p 之間的親合力與催化反應的最大速率。
此外，在ALD6 DNA 序列鑑定分析結果顯示酵母菌菌株 YPH499 Genomic DNA 在2 次獨立的PCR 反應發現皆有5 個相同的突變位置，推測具有單核苷酸多態性 (SNP) 的可能性。為了證明ALD6 之多型性，使用另一種酵母菌菌株 BY4742 的Genomic DNA 進行PCR 反應，經定序結果顯示在YPH499 的突變位置並沒有在 BY4742 出現。因這五個變異皆為啞突變，所以酵母菌ALD6 基因仍是種高度保留序列之DNA。

關鍵字

酵母菌ALD6 基因 ； 單核苷酸多態性 ； Ald6p 純化 ； 酵素動力

並列摘要

Since 1950s，Yeast acetaldehyde dehyrogenase (ALDH) has been studied for many years. It is a key enzyme of the pyruvate dehydrogenase (PDH) bypass。Recently, the role of the five members of ALDH family (ALD genes) was found to be the essential task of generating acetyl-CoA in the cytosol. Based on literature, this ALDH is deduced on the left arm of chromosome XVI of Saccharomyces cerevisiae .
In our studies, we amplified cloning the ALD6 gene by polymerase chain recitation (PCR)。The size of this DNA fragment is 1,506bp,containing a restriction enzyme ( NdeI) reaction site in DNA 5’-terminal.The ALD6 gene was cloned to to pGEM®-T easy vector by TA-Cloning. Through plasmid transformation to E.coli DH5α, we obtained the pGEM-T-ALD6 plasmid DNA in preparative amount. We can then get a specific ALD6 gene fragment by NdeI、SpeI restriction enzymes from pGEM-T-ALD6. Expression vector pET-43.1c(+) was used to contruct pET-43-ALD6 by inserting ALD6 gene fragment to the（NdeI、SpeI）restriction enzyme reaction sites on pET-43.1c(+).
Mini preparation of pET-43-ALD6 plasmid DNA was obtained by transformation to E.coli strain DH5α, then transformed into four strains of E.coli expression hosts: BL21(DE3)、BL21pLysS、Rosetta(DE3)、BL21-CodonPlus(DE3)-RIL）, We used different inducer agents, incubation times and temperature, and enzyme activity assay to determined the optimal growth conditions. The results show：At 30℃,lactose induced Rosetta(DE3) is the best of the four strains tested . The optimal time 12 hr after induction.The reaction rate and induced amount of Ald6p recombinant protein are also optimal under these conditions.
We then used the optimal conditions for large-scale preparation of Ald6p followed by purification.The following resins (Amersham Biosciences company) are used for purification：1. Q Sepharose fast flow 2. Blue Sepharose 6 fast flow. Purified Ald6p was characterized on different aldehydes (Propionaldehyde,4-aminobutyraldehyde, 3-aminopropionaldehyde, betaine aldehyde, etc.）. km,Vmax values were determined in order to understand the affinity and highest reaction rates with different aldehydes.
In addition, from two independent PCRs using yeast strain YPH499 genomic as template, we found the ALD6 DNA sequence has five SNP sites by DNA sequence analysis. To verify, yeast strain BY4742 genomic DNA was used for PCR reaction. No SNPs were detected in BY4742. Since all these five SNPs are silent mutations, the ALD6 gene sequence is highly conserved in Yeast

並列關鍵字

Aldehyde dehydrogenase ； Yeast Ald6p ； ALD6 gene ； Single nulotide polymorphism (SNP)

參考文獻

1. Black, S. Yeast aldehyde dehydrogenase. Arch. Biochem. Biophys. 34,86–97 (1951).

2.Black, S. Potassium-activated yeast aldehyde dehydrogenase.Methods Enzymol. 1, 508–511 (1993).

7. Jacobson, M. K., and C. Bernofsky, Mitochondrial acetaldehyde dehydrogenase from Saccharomyces cerevisiae. Biochim. Biophys.Acta. 350, 277–291 (1974).

8. Tessier, W. D., P. G. Meaden, F. M. Dickinson and M. Midgley.Identification and disruption of the gene encoding the K+- activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 164, 29–34 (1998).

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