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

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. Supplementary Information The online version of this article (10.1007/s10858-020-00354-6) contains supplementary material, which is available to authorized users.


Figure S1
. Quantification of TS-β1AR E. coli expression and purification by gel electrophoresis and western blotting.Western blotting against the histidine tag was used for quantifying the early steps of membrane solubilization and purification (panel A), in which the TS-β1AR band is not distinguishable due to the presence of other proteins.For the later steps (after nickel affinity chromatography), protein bands were quantified from an SDS-page gel (panel B).Details of the purification steps, sample preparation, and quantification using the program ImageJ are described in Materials and Methods.The labels indicate: "not induced": E.coli cells before induction of protein expression, "induced": E.coli cells after induction of protein expression by addition of IPTG, "supernatant": supernatant of cell harvesting step, "insoluble urea": insoluble fraction after membrane solubilization resuspended with buffer containing 7M urea, "solubilized membrane": membrane fraction solubilized with detergents (DDM, CHAPS and CHS), "NiNTA FT": flow through from nickel column, "NiNTA W1-W5": wash fractions from nickel column, "PreSc, 4h and 12h": Cleavage reaction of fusion protein with 3C PreScission protease after 4 and 12 h, respectively, "SPFF FT": flow through from the fast flow cation exchange column (SPFF), "SPFF W": wash fraction from SPFF column, "SPFF top peak": center fraction of the absorption (280 nm) peak from the SPFF column, "empty": empty lane, "SPFF full peak": pool of all fractions of the TS-β1AR peak in the SPFF, "ALAC FT": flow through from the alprenolol -3 -affinity column (ALAC), "ALAC W": wash fraction from ALAC column, "ALAC elution": pool of fractions within the peak corresponding to the TS-β1AR in the ALAC, "SPHP FT": flow through from the high-performance cation exchange column (SPHP), "SPHP top peak": fraction with the higher absorption (280 nm) from SPHP column, "SPHP full peak": pool of fractions within the peak corresponding to the TS-β1AR in the SPHP column.
The western blot of the 3C reaction sample (panel A) shows the 3C PreScission protease and the cleaved thioredoxin (identified in the figure), but not the maltose-binding protein or the TS-β1AR bands because these two proteins do not carry a poly-histidine tag.The band of the fusion protein after the nickel affinity purification is marked by a green rectangle.The band for the TS-β1AR monomer in the SDS-page gel (panel B) after the last purification step is marked by a blue rectangle.Very small amounts of higher TS-β1AR oligomers are also visible in the concentrated 'top peak' fraction.
The elution profile of the 280 nm absorption of TS-β1AR is shown in blue.The center of the TS-

β1AR
peak elutes at a conductivity (red line) of about 40 mS/cm.SDS-page analyses of the 'full peak' and 'top peak' from the SPHP elution are shown in panel B.

Figure S2 .
Figure S2.Comparison of 1 H-15 N TROSY spectra of 15 N-valine-labeled TS-b1AR obtained from E. coli (blue) or insect cells (red) in complex with the antagonist cyanopindolol.Additional peaks and a relative weakening of valine resonances in the E. coli receptor spectra are the result of efficient metabolic scrambling in E. coli to alanine, isoleucine, leucine, glutamate and other branched amino acids.Both spectra were recorded for ~45 hours at 304 K on samples containing 165 µM (E.coli) or 150 µM (insect cell) TS-b1AR, 1 mM cyanopindolol, 20 mM Tris, 1 mM EDTA, 100 mM NaCl, 37 mM DM, 0.02% NaN3, 5% D2O, pH 7.5.