Cloning And Expression Of Alcohol Dehydrogense Gene And Identification Of Its Biologic Activity

Human alcohol dehydrogenase (ADH, EC 1.1.1.1) is a cytosolic, dimeric, zinc-containing and NAD-dependent enzyme. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones, and acts as rate-limiting enzyme in ethanol metabolism. The new nomenclature organizes seven human alcohol dehydrogenase genes into five classes. It has the characteristics of gene polymorphism, ethnic specificity, gender specificity, tissue specificity and time specificity. Recently there have been many researches on its kinetic property, structure and function, but the overall mechanism for the entire alcohol dehydrogenase system has as yet to be established.Little amount of alcohol is beneficial. However, excessive alcohol consumption could induce alcohol toxicity, showing rubicundity, quick heartbeat, low blood pressure, swirl, surfeit, vomit and et al. It could even cause organ pathologic changes such as liver cirrhosis, digestive tract cancer, heart disease and so on. With the increasing consumption of alcohol, people are increasingly demand for anti-alcohol and liver-protection materials. Human ADH drug is superior to other congener products due to its source and side-effect. So applying of alcohol dehydrogenase on developing anti-alcohol drugs will be its important application and future development trend. Because isoenzyme ADH1B2 has the highest activity in human ADH class, its study on gene engineering may have great economic and social values.In this paper, ADH1B2 gene was obtained by RT-PCR using human liver total RNA as template and was cloned into pUCm-T vector. Then according to restrict site on primers, we insert ADH1B2 gene into pProEX HTb vector to construct plasmid pProEX-ADH1B2. ADH1B2 and 6×His were fused-expression in E.coli BL21(DE3). After perfect expression in 37℃environment inducted by 0.5mM IPTG for 7h, high-yield expression of ADH1B2 fused protein, existing in the inclusion body, was achieved, and it is 58% of total bacteria protein. This data exceeds any other prokaryotic expression system before. The washed inclusion body achieved high purity and this is useful for industrialized production. Nickel affinity chromatography was used to obtain higher purity protein according to its 6×His tag. The results indicated that target protein was firstly eluted by 60 mmol/L pyrazole and then reached elution peak by 100 mmol/L pyrazole. The purified protein was refolded by dialyzing in refolding solution with L-arginine. The results showed that the refolded protein has ADH activity through tracing A340. We are first to express ADH1B2 protein using prokaryotic expression vector pProEX HTb.