醛酮还原酶AKR7A5蛋白的纯化及其对萘醌类化合物的底物特异性研究  

Purification of recombinant AKR7A5 protein and measurement of substrate specificity of AKR7A5 towards naphthoquinone and its derivatives

作　　者：李丹[1] 刘雅茹[1] 张岐山[1] 初阳[2] 

机构地区：[1]中国医科大学药学院,辽宁沈阳110001 [2]中国医科大学附属第一医院药学部,辽宁沈阳110001

出　　处：《细胞与分子免疫学杂志》2012年第8期825-827,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：沈阳市科学技术计划(1091142-9-00);辽宁省自然科学基金(21012295)

摘　　要：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。AIM： To investigate the substrate specificity of mouse aldo-keto reductase AKR7A5 protein towards naphthoquinone and its derivatives.METHODS： The recombinant His-tagged AKR7A5 fusion protein in E.coli BL21pLysS cell strain was induced by IPTG and purified using FPLC system through HiTrap affinity column.The purified recombinant AKR7A5 protein was confirmed by SDS-PAGE and Western blotting.AKR enzyme assay was applied to measure the substrate specificity of recombinant AKR7A5 protein towards naphthoquinone and its derivatives.RESULTS： Recombinant His-AKR7A5 was successfully purified as confirmed by SDS-PAGE and Western blotting.AKR enzyme assay indicated that the recombinant AKR7A5 protein exhibited mild substrate specificity towards lawsone and low specificity towards juglone and vitamine K3,but no activity towards 1,4-naphthoquinone.CONCLUSION： AKR7A5 has selective substrate specificity towards naphthoquinone derivatives,suggesting that the aldo-keto reductase could play an important role in metabolism of certain naphthoquinone derivatives.

关 键 词：醛酮还原酶 萘醌类化合物 蛋白纯化 底物特异性 

分 类 号：R392.1[医药卫生—免疫学] Q55[医药卫生—基础医学]