Expression of the human soluble epoxide hydrolase in Escherichia coli by auto-induction for the study of high-throughput inhibition assays.
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.
Nishi
Kosuke
K
Industry and Academic Cooperation Laboratory for Drug Development, Mokpo National University, Muan, Jeonnam 534-729, South Korea. kosuke.nishi@tecan.com
Kim
In-Hae
IH
Ma
Seung-Jin
SJ
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007313:Insecta; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
D004791:Enzyme Inhibitors; D004851:Epoxide Hydrolases