Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Genetics and Molecular Biology

Nan Wang¹  Haifeng Shi¹  Qin Yao¹  Yang Zhou¹  Lequn Kang¹  Huiqin Chen¹  Keping Chen¹ 

[1] ,Jiangsu University, Institute of Life Sciences Jiangsu Province,China

关键词: 5'-RACE PCRADH;    enzymatic activity;    recombinant protein;   

DOI  :  10.1590/S1415-47572011000200013

来源: SciELO

【 摘 要 】

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.

【 授权许可】

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