Protein Expression in E. coli

Protein expression in E. coli was done as previously described (Budaitis et al., 2021 (link)). Briefly, a plasmid was transformed into BL21-CodonPlus(DE3)-RIPL-competent cells (Agilent #230280), and a single colony was inoculated in 1 ml of TB with 50 µg/ml of chloramphenicol and 25 µg/ml of carbenicillin or 15 µg/ml of kanamycin in the case of Sfp. The culture was shaken at 37°C overnight, and then inoculated into 400 ml of TB, which was shaken at 37°C for 5 hr, and subsequently cooled down to 18°C. IPTG was added to the culture to a final concentration of 0.1 mM, and the expression was induced overnight at 18°C with shaking. The culture was harvested by centrifugation at 3000 rcf for 10 min. Following the removal of the supernatant, the cell pellet was resuspended in 5 ml of B-PER complete (Thermo Scientific #89821) supplemented with 4 mM MgCl₂, 2 mM EGTA, 0.2 mM ATP, 2 mM DTT, and 2 mM PMSF. The cell suspension was then flash-frozen and stored at –80°C.