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High-level soluble expression of one model olfactory receptor (ODR-10) in Escherichia coli cell-free system

Author:
Zhang, Xu, Sheng, Jiayuan, Huang, Lei, Du, Liping, Cai, Jin, Cen, Peilin, Xu, Zhinan
Source:
World journal of microbiology & biotechnology 2014 v.30 no.3 pp. 893-901
ISSN:
0959-3993
Subject:
Caenorhabditis elegans, Escherichia coli, cell free system, cytotoxicity, detergents, hydrophobicity, membrane proteins, odor compounds, receptors
Abstract:
High-level production of G protein-coupled receptors (GPCRs) is usually difficult to achieve in heterologous cell systems. The inherent hydrophobicity of these receptors could cause aggregation and possible cytotoxicity. Cell-free (CF) expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. Here we reported the CF production of an olfactory receptor from Caenorhabditis elegans, odorant response abnormal protein 10 (ODR-10), a member of GPCRs, using the Escherichia coli extracts. Different expression vectors were investigated and 175 μg/ml total ODR-10 was achieved with pIVEX2.4c. To obtain soluble ODR-10, different detergents and liposome with varied concentrations were respectively added into the CF system. High-level expression of soluble ODR-10 (150 μg/ml) was attained with the addition of 1.5 % polyoxyethylene-(20)-cetyl-ether (Brij58) into the CF system. Furthermore, the yield of total ODR-10 was improved to 350 μg/ml by supplementing liposomes into the CF system, and the maximal concentration of the soluble receptor (102 μg/ml) was achieved in this liposome-assisted CF system. Both strategies produced ODR-10 efficiently by using CF system, and the direct reconstitution of the in vitro expressed receptor into liposomes will be preferred for its potential applications in many areas.
Agid:
621017