[Cloning, expression and evaluation of Saccharomyces cerevisiae ADH2].
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.
Yu
Mengbin
M
Laboratory of Biology Defense, Command and Engineering College of Chemical Defense, Beijing, China.
Zhi
Qingwen
Q
Xu
Li
L
Zhao
Chuangxin
C
Chen
Gaoyun
G
Jiang
Yongchao
Y
Liu
Min
M
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D051379:Mice; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C509911:ADH2 protein, S cerevisiae; D000426:Alcohol Dehydrogenase