Nishi Kosuke, Kim In-Hae, Ma Seung-Jin
Industry and Academic Cooperation Laboratory for Drug Development, Mokpo National University, Muan, Jeonnam 534-729, South Korea.
Protein Expr Purif. 2010 Jan;69(1):34-8. doi: 10.1016/j.pep.2009.09.013. Epub 2009 Sep 25.
Soluble epoxide hydrolase (sEH) is a key enzyme involved in the metabolism of epoxy fatty acid mediators such as epoxyeicosatrienoic acids with emerging roles in the regulations of hypertension and inflammation. Inhibitors of human sEH (hsEH) are effective drug candidates for the treatment of cardiovascular diseases. Preparation of hsEH for enzyme inhibition studies has been carried out by using baculovirus expression system. We herein explored the feasibility of expression of hsEH in Escherichia coli cells for the study of high-throughput screening assays of enzyme inhibitors, because the bacterial expression system is easier to handle and more cost-effective than the baculovirus expression system. The functional target enzyme was successfully produced in prokaryotic expression system by an auto-induction method and exhibited comparable enzyme activity to that yielded in baculovirus expression system. The bacterial-hsEH showed similar sensitivity to the baculovirus-hsEH against six reported inhibitors. Overalls indicate that bacterial expression of hsEH employed in the present study is useful for preparing enzymatically active hsEH, leading to effective performance of high-throughput screening assay of hsEH inhibitors and to rapid identification of novel drug candidates for the treatment of cardiovascular diseases.
可溶性环氧化物水解酶（sEH）是参与环氧脂肪酸介质（如环氧二十碳三烯酸）代谢的关键酶，在高血压和炎症调节中发挥着新作用。人sEH（hsEH）抑制剂是治疗心血管疾病的有效候选药物。利用杆状病毒表达系统进行了用于酶抑制研究的hsEH的制备。我们在此探讨了在大肠杆菌细胞中表达hsEH用于酶抑制剂高通量筛选测定研究的可行性，因为细菌表达系统比杆状病毒表达系统更易于操作且成本效益更高。通过自动诱导方法在原核表达系统中成功产生了功能性靶酶，其表现出与杆状病毒表达系统中产生的酶活性相当的酶活性。细菌hsEH对六种已报道的抑制剂显示出与杆状病毒hsEH相似的敏感性。总体而言，本研究中采用的hsEH细菌表达对于制备具有酶活性的hsEH是有用的，从而能够有效地进行hsEH抑制剂的高通量筛选测定，并快速鉴定用于治疗心血管疾病的新型候选药物。
