Design of an engineered Gα_q protein for high-yield expression and NMR isotope labeling in E. coli

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets that signal through heterotrimeric G proteins. While structural insights into Gα_s and Gα_i/o subfamilies have advanced significantly, characterizing the Gα_q/11 subfamily remains challenging due to poor soluble expression in prokaryotic systems, precluding high-yield isotope labeling needed for NMR studies. Here, we present an efficient expression strategy for Gα_q protein in Escherichia coli. We identified the H5 helix as a critical determinant of folding and solubility. By swapping this helix with its Gα_i/o counterpart and employing low-temperature co-expression with bacterial chaperones, we significantly enhance soluble yields while retaining GTPase activity. The ¹⁵N-labeled protein exhibited well-dispersed ¹H-¹⁵N HSQC spectra, validating the suitability of this expression strategy for future high-resolution NMR investigations.