Ribosome purification

For 5 g of cells, the pellet was re-suspended in 30 ml of buffer A (20 mM HEPES-KOH pH 7.5, 100 mM NH₄Cl, 21 mM Mg(OAc)₂, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT), supplemented with the addition of 600 μl of protease inhibitor cocktail (one tablet (Roche), dissolved in 1 ml buffer A), of 300U DNase I (Roche) and of 3.5 mg lysostaphin (Sigma-Aldrich), before being lysed at 37°C for 45 min. Cell debris were removed by centrifugation at 30 000 ×g for 90 min.

The resulting supernatant was further subjected to a differential precipitation by PEG, similarly to the protocol employed for Saccharomyces cerevisiae (58). PEG 20 000 was added from a 30% w/v stock solution (Hampton Research) to a final concentration of 2.8% w/v for the first fractionation. The solution was then centrifuged at 20 000 ×g for 5 min. The supernatant was recovered and PEG 20 000 was increased to 4.2% w/v for the second fractionation. The solution was then centrifuged at 20 000 ×g for 10 min, and the ribosome pellet was resuspended in 35 ml buffer A and layered on 25 ml of a sucrose cushion (10 mM Hepes-KOH pH 7.5, 500 mM KCl, 25 mM Mg(OAc)₂, 1.1 M Sucrose, 0.5 mM EDTA, 1 mM DTT). Centrifugation was subsequently carried out at 45 000 rpm for 15 h using a Beckman Type 45 Ti rotor.

The crude ribosome pellet was resuspended in buffer E (10 mM Hepes-KOH pH 7.5, 100 mM KCl, 10 mM Mg(OAc)₂, 0.5 mM EDTA, 1 mM DTT) up to a concentration of 7 mg.ml⁻¹. Exactly 0.5 ml were loaded on 7–30% sucrose density gradients and centrifuged at 17 100 rpm for 15.5 h using a Beckman SW28 rotor. The fractions corresponding to 70S particles were pooled, the concentration of Mg(OAc)₂ was adjusted to 25 mM and PEG 20 000 was added to a final concentration of 4.5% w/v. Ribosomes were pelleted by centrifugation at 20 000 ×g for 12 min, the pellet was gently dissolved in buffer G (5 mM Hepes-KOH pH 7.5, 50 mM KCl, 10 mM NH₄Cl, 10 mM Mg(OAc)₂, 1 mM DTT) to a final concentration of 20–25 mg.ml⁻¹. Aliquots of 30 μl were flash frozen in liquid nitrogen and stored at −80°C. A typical yield was 10–12 mg of ribosomes from 5 g of cells.