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

• Main Authors: Yu-Chun Chen, 陳煜群
• Other Authors: Ming-Kai Chern
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/56197368418250446865

碩士 === 淡江大學 === 生命科學研究所碩士班 === 93 === 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