Expression and Purification of Rhodopsin-Gi Complex

The N2C/M257Y/D282C mutant of bovine rhodopsin was expressed as described (Deupi et al., 2012 (link)) in human embryonic kidney (HEK) 293 GnTI-deficient cells (gift from Philip Reeves), which retain all post-translational modifications of the native protein including palmitoylation of Cys residues at positions 322 and 323 (Standfuss et al., 2011 (link)). Cell lines were tested and confirmed to be mycoplasma-free. The human Gαi subunit (Gαi1) with an N-terminal TEV protease-cleavable deca-histidine tag was expressed in the E. coli BL21 (DE3) strain (Sigma: CMC0014) and purified as described (Sun et al., 2015 (link)). The transducin heterotrimer was isolated from the rod outer segment of bovine retina (W L Lawson Company) and Gβ1γ1 was separated from Gαt with Blue Sepharose 6 Fast Flow (GE Healthcare) as described (Maeda et al., 2014 (link)). The Gαi1β1γ1 heterotrimer (Gi) was prepared by mixing equimolar amounts of Gαi1 with or without 10xHis-tag and Gβ1γ1 and incubated at 4°C for 1 hr shortly before use for rhodopsin-Gi complex formation on the 1D4 immunoaffinity column.