Efficient production of a functional G protein-coupled receptor in E. coli for structural studies

Abiko, Layara Akemi and Rogowski, Marco and Gautier, Antoine and Schertler, Gebhard and Grzesiek, Stephan. (2021) Efficient production of a functional G protein-coupled receptor in E. coli for structural studies. Journal of Biomolecular NMR, 75 (1). pp. 25-38.

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G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey β 1 -adrenergic receptor (β 1 AR) uniformly or selectively labeled in 15 N or 2 H, 15 N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2-0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β 1 AR mutant also comprises the two native tyrosines Y 5.58 and Y 7.53 , which enable G protein binding. High-quality 1 H- 15 N TROSY spectra were obtained for E. coli -expressed YY-β 1 AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
UniBasel Contributors:Grzesiek, Stephan and Abiko, Layara Akemi and Rogowski, Marco
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:16 Feb 2023 07:58
Deposited On:30 Apr 2021 13:07

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