Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain

Abstract

The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs' conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties in vivo. Thus far, the preparation of the recombinant enzyme for enzymatic and structural in vitro studies has been performed mainly using a baculovirus expression system. More recently, it was reported that the enzyme could be exogenously expressed and isolated from E. coli, although limited amounts of active protein were obtained. We herein describe two novel methods to yield pure recombinant enzyme. The first describes the expression and purification of the full-length enzyme from eukaryotic cells HEK293-F, whilst the second concerns the C-terminal domain of hsEH obtained from the cost-effective and rapid E. coli prokaryotic system. The two methods successfully generated satisfactory amounts of functional enzyme, with virtually identical enzymatic activity. Overall, the protocols described in this paper can be employed for the recombinant expression and purification of active hsEH, to be used in future biomedical investigations and for high-throughput screening of inhibitors for potential use in the treatment of cardiovascular disease.

15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism

“…To purify the covalent adducts, a bespoke separation strategy via Benzylthio-Sepharose (BTS) affinity chromatography resin was devised. BTS is able to bind sEH through interactions between the enzyme’s hydrophobic active site and benzylmercaptan moieties immobilised onto Sepharose beads 40,41 . We hypothesised that the modification by 15d-PGJ 2 might affect this interaction, possibly through conformational effects (see below).…”

“…The EPHX2 (T230-M555—hsEH CTD) cDNA was cloned in the bacterial expression vector pET3a, as described previously 41 . Single mutants C423S and C522S were generated with the Q5 ® Site-Directed Mutagenesis Kit (NEB).…”

“…The hsEH CTD and its mutants were expressed in Ros2 TM (DE3) (EMD Millipore) and purified as described previously for WT 41 .…”

“…To purify the covalent adducts, we designed a tailored Benzylthio-Sepharose (BTS) affinity chromatography protocol. Apo-hsEH CTD binds BTS via interactions between the enzyme’s hydrophobic binding pocket and benzylmercaptan moieties immobilised onto Sepharose beads 40,41 , whilst covalently adducted counterparts do not interact with the resin and were collected in the column flow-through fractions. 15d-PGJ 2 -hsEH CTD covalent adducts were prepared as follows.…”

“…0.5 mg of WT, C423S, C522S, and C423S/C522S proteins were dialysed overnight at 4 °C in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.4, 75 mM NaCl, 5% glycerol, 10 μM TCEP (binding buffer), and then incubated at 4 °C 1:8 v/v (molar ratio) with 15d-PGJ 2 (dissolved in PBS) or binding buffer (as control). After overnight incubation, the mixtures were loaded onto 500 μL of BTS resin in gravity columns (prepared in-house 41 ), previously equilibrated in binding buffer. After 15 min incubation at room temperature, the flow-through was collected and the resin washed five times with 500 μL of binding buffer.…”