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

Research output: Contribution to journalArticlepeer-review

Original languageEnglish
Pages (from-to)105-113
Number of pages9
JournalProtein expression and purification
Volume153
Early online date12 Sep 2018
DOIs
Accepted/In press5 Sep 2018
E-pub ahead of print12 Sep 2018
PublishedJan 2019

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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 inhibitors screenings, to effectively identify drug candidates for the treatment of cardiovascular diseases.

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