Purification of 30S Ribosomal Subunits

E6006 bacteria were grown at 30 °C in M63 medium [46 ] with 10 mM glucose to an OD₆₀₀ of 0.2. The cells were then heated to 45 °C by adding medium that had been pre-warmed to 60 °C. The cultures were incubated for 1 h, then cooled to 4 °C on ice. Cells were collected by centrifugation at 8000× g for 10 min, with the resulting pellets re-suspended in buffer A (50 mM Tris-HCl pH 7.5, 15 mM Mg(C₂H₃O₂)₂, 100 mM NH₄Cl, 3 mM HOCH₂CH₂SH, 0.5 mM EDTA, and 1 mM PMSF) and disrupted using a French press (1250 PSI). The lysate was centrifuged at 15,000× g for 30 min at 4 °C. The supernatant (250 A₂₆₀ units) was centrifuged on a 38-mL (10–40%) sucrose gradient using an SW 28 rotor working at 21,000 rpm for 17 h at 4 °C. Fractions of interest were then pooled from different gradient tubes and concentrated via centrifugation at 100,000× g for 6 h. Pellets were dissolved in buffer A then analyzed using IMAC chromatography with Ni-NTA resin (Qiagen, France). All 70S ribosomes and 30S fractions containing his-tagged uS2 proteins were eliminated by three successive Ni-NTA purification steps. Particles retained by the resin were eluted with 100 mM imidazole in buffer A. The effluent underwent chromatography on Strep-Tactin sepharose (IBA, Göttingen, Germany) to bind particles containing the bS20 strep protein. These particles were eluted with 2.5 mM desthiobiotin in buffer A, concentrated with Amicon (100MCW) cartridges, and then analyzed by SDS-PAGE electrophoresis. The purification of mature 30S subunits from E6006 cells was done in the same way as described here for the precursors.