Expression of the human soluble epoxide hydrolase in Escherichia coli by auto-induction for the study of high-throughput inhibition assays Kosuke Nishi a, * , In-Hae Kim a , Seung-Jin Ma b a Industry and Academic Cooperation Laboratory for Drug Development, Mokpo National University, Muan, Jeonnam 534-729, South Korea b Department of Food Engineering, Mokpo National University, Muan, Jeonnam 534-729, South Korea article info Article history: Received 28 May 2009 and in revised form 16 September 2009 Available online 25 September 2009 Keywords: Soluble epoxide hydrolase Escherichia coli Baculovirus Screening Enzyme inhibitor abstract Soluble epoxide hydrolase (sEH) is a key enzyme involved in the metabolism of epoxy fatty acid media- tors such as epoxyeicosatrienoic acids with emerging roles in the regulations of hypertension and inflam- mation. 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 bacte- rial-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 enzymat- ically 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. Ó 2009 Elsevier Inc. All rights reserved. Introduction Mammalian soluble epoxide hydrolase (sEH; EC 3.3.3.2) is a homodimer consisting of two domains; The C-terminal domain of sEH plays a role in catalyzing epoxide hydrolysis, whereas the N- terminal domain has a phosphatase activity [1,2]. The sEH has been demonstrated to hydrate endogenous epoxy fatty acid mediators such as epoxyeicosatrienoic acids (EETs) 1 involved in the regulation of hypertension, inflammation, and other cardiovascular diseases [3– 5]. The hydrated products of EETs catalyzed by sEH are believed to have dramatically reduced biological activities, implying that main- taining EET concentrations in vivo through the sEH inhibition is highly effective for treating some disorders. Urea- or amide-based compounds substituted with various alkyl groups exhibited highly potent inhibitions for human sEH (hsEH), resulting in significant bio- logical effects in disease animal models [6–8]. Recently, a few inhib- itors screened out from a large number of compounds for inhibition potency on hsEH have been filed as investigational new drug candi- dates to treat the diseases, suggesting that building up a variety of chemically diverse libraries and finding out promising lead struc- tures will be highly valuable for developing novel drug candidates. Rapid, cost-effective identification of such novel lead com- pounds is driven from simple and easy-handling preparation of functional target proteins. In general, heterologous expression in Escherichia coli cells is chosen in priority to produce target recom- binant human enzymes, because the bacterial expression system is easy to engineer, practical and immediate to obtain target proteins. However, screening of hsEH inhibitors have been carried out by using hsEH produced from insect cells via baculovirus expression system [9–11], which is generally much more time-consuming and laborious than the prokaryotic expression system. No reports describing the discovery of hsEH inhibitors using the enzyme ex- pressed in E. coli cells have been published. Because baculovirus expression system is highly effective to produce recombinant hu- man enzymes with an appropriate folding leading to maintenance of enzyme activity while the prokaryotic expression system often fails to produce active recombinant human enzymes, it is assumed that production of enzymatically active hsEH is of great difficulty like an example of a rat liver carboxylesterase that showed the bac- ulovirus expression is suitable to produce the active enzyme while bacterial expression system induces inactive form of the enzyme [12]. On the other hand, it was reported that recombinant rat sEH was successfully produced in E. coli cells and used for measur- ing enzyme activities with epoxide substrates [13,14]. Also, recom- 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.09.013 * Corresponding author. Present address: Tecan Japan Co., Ltd., 580-16 Horikawa- cho, Saiwai-ku, Kawasaki 212-0013, Japan. Fax: +81 44 556 7312. E-mail address: kosuke.nishi@tecan.com (K. Nishi). 1 Abbreviations used: EETs, epoxyeicosatrienoic acids; sEH, soluble epoxide hydro- lase; hsEH, human sEH; IPTG, isopropyl-b-D-thiogalactopyranoside; CMNPC, cyano- (2-methoxynaphthalen-6-yl)-methyl trans-(3-phenyl-oxyran-2-yl)-methyl carbon- ate; TEV, tobacco etch virus. Protein Expression and Purification 69 (2010) 34–38 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep