Expression and Purification of His-Tagged Human GNA1

The expression plasmid for human GNA1 with N-terminal His₆-tag was cloned previously (Ruegenberg et al., 2020 (link)). Human GNA1 with N-terminal His₆-tag was expressed in Rosetta (DE3) E. coli cells. LB cultures were incubated at 37°C and 180 rpm until an OD₆₀₀ of 0.4–0.6 was reached. Then, protein expression was induced by addition of 0.5 mM IPTG and incubated for 3 hr at 37°C and 180 rpm. Cultures were harvested and pellets were stored at –80°C. Human GNA1 purification protocol was adopted from Hurtado-Guerrero et al., 2008 (link) with small modifications. E. coli were lysed in 50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 10 mM imidazole, 2 mM 2-mercaptoethanol, 5% (v/v) glycerol with complete EDTA-free protease inhibitor cocktail (Roche), and 10 µg/ml DNAseI (Sigma-Aldrich) by sonication. The lysate was clarified by centrifugation and the supernatant was loaded on Ni-NTA Superflow affinity resin (Qiagen). The resin was washed with wash buffer (50 mM HEPES/NaOH pH 7.2, 500 mM NaCl, 50 mM imidazole, and 5% (v/v) glycerol) and the protein was eluted with wash buffer containing 250 mM imidazole. Eluted protein was then dialyzed against storage buffer (20 mM HEPES/NaOH pH 7.2, 500 mM NaCl, and 5% (v/v) glycerol).