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

Autor: Abis G; Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Excellence, School of Basic and Medical Biosciences, King's College London, London, SE1 1UL, United Kingdom. Electronic address: giancarlo.abis@kcl.ac.uk., Charles RL; Cardiovascular Division and British Heart Foundation Centre of Excellence, The Rayne Institute, King´s College London, St Thomas' Hospital, London, SE1 7EH, United Kingdom., Eaton P; Cardiovascular Division and British Heart Foundation Centre of Excellence, The Rayne Institute, King´s College London, St Thomas' Hospital, London, SE1 7EH, United Kingdom., Conte MR; Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Excellence, School of Basic and Medical Biosciences, King's College London, London, SE1 1UL, United Kingdom. Electronic address: sasi.conte@kcl.ac.uk.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2019 Jan; Vol. 153, pp. 105-113. Date of Electronic Publication: 2018 Sep 12.
DOI: 10.1016/j.pep.2018.09.001
Abstrakt: 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.
(Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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