Table 1.
Solubility and maximum yields of GPCRs produced using cell-free in vitro translation in the presence of Brij-35.
Figure 1.
Detergent screen for cell-free GPCR production.
Detergents commonly used for membrane protein solubilization or crystallization were screened. Figure 1 shows a detergent screen with the receptor hOR17-210. Brij-35 and Brij-58 yielded ∼4–5 times as much receptor as the next best detergent. Although comparable, Brij-35 consistently had slightly higher yields than Brij-58. Each bar represents the average of 2–3 experiments. The data was normalized to Brij-35.
Figure 2.
Silver Stains of Purified GPCRs.
A) Four cell-free expressed GPCRs. B) Comparison between cell-free and HEK293 expressed hVN1R1. Most GPCRs could be purified to >90% purity, and all showed two bands characteristic of a monomer and a dimer [17], [18]. The cell-free and HEK293 expressed receptors run at the same size, and have similar purities.
Figure 3.
Circular Dichroism Spectra of Five Purified GPCRs.
A) Cell-free expressed mOR103-15 made with Brij-35 or no detergent, B) Cell-free expressed hTAAR5, C) Cell-free expressed hFPR3, and D) Cell-free and HEK293 expressed hVN1R1. All purified GPCRs have characteristic alphahelical spectra, except mOR103-15 made without detergent. Since GPCRs have 7-transmembrane helices, and an overall α-helix content of ∼50%, the CD spectra suggest that these receptors are properly folded. The near overlap of the spectra for cell-free and HEK293 expressed hVN1R1 suggests that both receptors are properly folded, and further indicates that cell-free produced GPCRs are comparable to those expressed in mammalian cells.
Figure 4.
Microscale Thermophoresis Measurements of Purified GPCRs.
A) Cell-free expressed hVN1R1 with and without heat-denaturation. B) HEK293 expressed hVN1R1 with and without heat-denaturation. The non-denatured receptors show typical sigmoidal binding curves, with plateaus at low and high concentrations. Cell-free expressed hVN1R1 has an EC50 of 6±2 µM, and HEK293 expressed hVN1R1 has an EC50 of 3.5±0.7 µM. The heat-denatured controls had flat responses or random amplitudes throughout the ligand titration range. These results show that hVN1R1 is binding carveol. Furthermore, the similar EC50 values and binding curves in A) and B) demonstrate that cell-free produced receptors function as well as HEK293 expressed receptors. The curves were normalized to the fraction of bound receptor. Each data point represents the mean of 3 independent experiments; error bars show the standard deviation. The binding curves were fit to the Hill equation. The binding results shown are representative of the data from other binding measurements.