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目的:探讨小鼠醛酮还原酶AKR7A5蛋白对萘醌及其衍生物的底物特异性。方法:IPTG(异丙基硫代半乳糖糖苷)诱导BL21pLysS大肠杆菌中His标签的AKR7A5融合蛋白大量表达,利用FPLC系统通过HiTrap亲和柱纯化His-AKR7A5融合蛋白,经SDS-PAGE和Western blot法鉴定纯化的AKR7A5蛋白。使用AKR酶活性实验检测纯化的重组AKR7A5蛋白对萘醌类化合物的底物特异性。结果:经SDS-PAGE和Western blot验证,成功纯化了His标签的AKR7A5融合蛋白;AKR酶活性实验结果显示,重组AKR7A5蛋白对散沫花醌有中等亲和力,对胡桃醌和维生素K3有较低的亲和力,对1,4-萘醌无亲和力。结论:成功纯化了重组AKR7A5蛋白,并检测了其对萘醌类化合物的底物特异性,结果表明醛酮还原酶很可能选择性地参与萘醌类化合物的代谢。
Objective: To investigate the substrate specificity of the mouse aldokinase reductase AKR7A5 protein to naphthoquinone and its derivatives. Methods: IPTG (isopropylthiogalactoside) was used to induce the expression of His-tagged AKR7A5 fusion protein in BL21pLysS E.coli. His-AKR7A5 fusion protein was purified by HiTrap affinity column using FPLC system. SDS-PAGE and Western blot Purified AKR7A5 protein was identified. The substrate specificity of the purified recombinant AKR7A5 protein for naphthoquinone compounds was tested using the AKR enzyme activity assay. Results: The His-tagged AKR7A5 fusion protein was successfully purified by SDS-PAGE and Western blot. The results of AKR enzyme activity showed that the recombinant AKR7A5 protein had moderate affinity for henhydroquinone and lower affinity for juglone and vitamin K3 Affinity, no affinity for 1,4-naphthoquinone. CONCLUSION: The recombinant AKR7A5 protein was successfully purified and its substrate specificity for naphthoquinone compounds was tested. The results showed that aldehyde-keto reductase is likely to participate selectively in the metabolism of naphthoquinone compounds.