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

Abstract

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 β1-adrenergic receptor (β1AR) uniformly or selectively labeled in 15N or 2H,15N. 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-β1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-β1AR 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.

Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol

“…CLR is highly abundant in human cells and essential for maintaining cell excitability and homeostasis [30][31][32][33][34][35][36][37][38] . CLR increases GPCR thermal stability [39][40][41][42][43][44][45] and often acts as an allosteric modulator of GPCR activity [46][47][48][49][50][51] . The direction of modulation can vary, as CLR e.g.…”

Quantitative analysis of sterol-modulated monomer–dimer equilibrium of the β 1 -adrenergic receptor by DEER spectroscopy

“…To investigate the effects of CHS and cholate on the structural preferences of β 1 AR by DEER, we made use of the naturally occurring cysteines present in a construct of thermostabilized turkey β 1 AR optimized for expression in Escherichia coli ( 33 ) (see Experimental Procedures for details of purification and reconstitution, and SI Appendix , Figs. S1 A and B ).…”

Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

“…Typically, the total exogenous yield of functional GPCRs confounds functional studies. Thus, GPCR production continues to be an area of innovation ( Abiko et al, 2021 ; Mulry et al, 2021 ). Here, we implemented two techniques, codon harmonization and an MBP fusion tag, to facilitate the production of a functional human GPCR from E. coli .…”