AI Article Synopsis
- Researchers cloned and expressed the ADH2 gene from the yeast Saccharomyces cerevisiae in E. coli BL21 (DE3) using total RNA and RT-PCR to create a recombinant plasmid.
- The recombinant plasmid pTAT-ADH2 was transformed into E. coli and induced with IPTG to produce the ADH2 enzyme, which was then purified and tested for activity.
- The sequence analysis showed 90% homology with the GenBank report, and the expressed ADH2 demonstrated significant ethanol dehydrogenase activity in biochemical and mouse toxicological tests.
Article Abstract
In order to clone and express alcohol dehydrogenase II (ADH2) gene from Saccharomyces cerevisiae in E. coli BL21 (DE3) efficiently, we extracted the total RNA as template and obtained ADH2 gene by RT-PCR and connected ADH2 gene to pTAT plasmids to gain recombinant expression plasmid pTAT-ADH2, then transformed this recombinant expression plasmid pTAT-ADH2 into E. coli BL21 (DE3). The recombinant was induced by IPTG to express ADH2. After purification, ADH2 activity was tested in vitro and toxicologic test was done in mouse. Sequence test showed that the acquired fragments exhibited 90% homology to ADH2 gene sequence from GenBank report. The target gene expressed efficiently and took up to approximant 50% of total protein by SDS-PAGE and band scanning analysis. The purified protein exhibited the identified activity through biochemical test and mouse toxicological test. As a result, the acquired ADH2 gene was highly homology to the published sequence and expressed at a high level in E. coli BL21 (DE3), more importantly, ADH2 proved to have ethanol dehydrogenase activity.
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