Q hiload column

Typically, 500 ml cultures of BL21-Rosetta cells overproducing Pa-aIF1 were harvested, resuspended in 20 ml of buffer A (10 mM HEPES pH 7.5, 3 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.1 mM PMSF, 0.1 mM benzamidine) plus 100 mM NaCl and disrupted by sonication. The crude extract was then heated 10 min at 70°C. After pelleting the non-thermostable proteins, the supernatant was loaded onto a Q-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer A plus 100 mM NaCl. The flow through was recovered and loaded onto an S-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer A plus 100 mM NaCl. Pa-aIF1 was eluted by applying a NaCl gradient (100–600 mM). The recovered sample was then concentrated and loaded onto a superdex 75 (HR 10/30, GE Healthcare). The purified protein was finally concentrated to ∼200 μM and stored at –20°C in buffer A with 55% glycerol for further use in toeprinting experiments (Supplementary Figure S1). Pa-aIF1 variants were purified using the same protocol adapted to the isoelectric points of the proteins and to their behaviors on the ion exchange columns.

Purification of Pa-30S and Pa-aIF1A were performed as described in (24 (link)). The untagged version of Pa-aIF2 was purified as follows. Cultures (250 ml) of cells overproducing each of the three subunits of Pa-aIF2 (32 (link)) were harvested, mixed in 40 ml of buffer B (500 mM NaCl, 10 mM HEPES pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.1 mM PMSF, 0.1 mM benzamidine) and disrupted by sonication. After centrifugation, the supernatant was heated for 10 min at 80°C. After pelleting, the supernatant was loaded onto a Q-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer B. The flow through was recovered and diluted two fold with buffer A. The sample was then loaded onto an S-Hiload column (10 mm × 4 cm; GE Healthcare) equilibrated in buffer A plus 250 mM NaCl. The assembled heterotrimer was eluted by applying a step of buffer A plus 700 mM NaCl. The recovered sample was then concentrated to 1 ml. The final heterotrimer preparation was obtained after purification by molecular sieving on a Superdex 200 HR column (10 mm × 30 cm, GE Healthcare) equilibrated in buffer B. Finally, glycerol (55% final concentration) and 1 mM Gpp(NH)p-Mg²⁺ were added. The protein was stored at –20°C for further use in toeprinting experiments. Routinely, 6 mg of purified protein were obtained from the three mixed 250 ml cultures.