Cloning, overexpression, purification and catalytic analysis of aldehyde dehydrogenase 2 and 3 from Saccharomyces cerevisiae
| Autor: | Ching-Yuan Mao, 毛清源 |
|---|---|
| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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