Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain.
Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH) and of its functional C-terminal domain.
复制标题
DOI：
10.1016/j.pep.2018.09.001
复制
发表时间：
2019-01
影响因子：
1.6
通讯作者：
Conte MR
中科院分区：
文献类型：
--
作者：
Abis G;Charles RL;Eaton P;Conte MR
人可溶性环氧化物水解酶（hsEH）是一种参与内源性抗炎和心脏保护信号传导介质（称为环氧二十碳三烯酸（Epoxyeicosatrienoic Acids，EHF））水解的酶。通过hsEH将雌二醇转化为相应的二醇产生非生物活性分子，因此预期酶抑制将增强雌二醇的生物利用度及其有益性质。已经开发了许多抑制剂来靶向该酶，其中一些在体内显示出有希望的抗高血压和抗炎特性。迄今为止，用于体外酶和结构研究的重组酶的制备主要使用杆状病毒表达系统进行。最近，有报道称该酶可以从大肠杆菌中获得外源表达和分离。大肠杆菌，虽然获得了有限量的活性蛋白。我们在此描述了两种新的方法来产生纯的重组酶。第一个描述了从真核细胞HEK 293-F中表达和纯化全长酶，而第二个涉及从成本有效和快速的E. coli原核表达系统。这两种方法成功地产生了令人满意的量的功能酶，几乎相同的酶活性。总体而言，本文中描述的协议可以用于重组表达和纯化的活性hsEH，用于未来的生物医学研究和高通量筛选的抑制剂用于治疗心血管疾病的潜在用途。hsEH是心血管稳态的关键调节剂。开发了hsEH全长（FL）的HEK 293-F哺乳动物表达系统。大肠建立了hsEH C-末端结构域（CTD）的原核表达系统。这两种蛋白在体外表现出相同的酶比活性。CTD制备提供了易于操作、高收率和高纯度的益处。
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. hsEH is a key regulator of cardiovascular homeostasis. A HEK293-F mammalian expression system for hsEH full-length (FL) was developed. An E. coli expression system for the hsEH C-terminal Domain (CTD) was established. Both proteins exhibited the same enzymatic specific activity in vitro. The CTD preparation provides benefits of easy operation, and high yield and purity.
登录
查看更多内容
DOI：
10.1016/j.bbrc.2012.02.108
发表时间：
2012-03-23
影响因子：
3.1
作者：
Morisseau C;Schebb NH;Dong H;Ulu A;Aronov PA;Hammock BD
通讯作者：
Hammock BD
影响因子：
6.1
作者：
GILL, SS;OTA, K;HAMMOCK, BD
通讯作者：
HAMMOCK, BD
影响因子：
2.9
作者：
Chen, Dawn;Whitcomb, Randall;Webb, Heather K.
通讯作者：
Webb, Heather K.
影响因子：
4.8
作者：
PINOT, F;GRANT, DF;HAMMOCK, BD
通讯作者：
HAMMOCK, BD
影响因子：
4.3
作者：
Hopmann, Kathrin H.;Himo, Fahmi
通讯作者：
Himo, Fahmi