A fresh O/N culture of E. coli JE28 was used to inoculate 1 l. LB with 50 µg/ml kanamycin and grown with aeration at 37°C. At A600 1.0, the culture was slowly cooled to 4°C to produce run-off ribosome and harvested by centrifugation at 4000 rpm for 30 min. The cell-pellet was resuspended in lysis buffer (20 mM Tris–HCl pH 7.6, 10 mM MgCl2, 150 mM KCl, 30 mM NH4Cl and PMSF protease inhibitor 200 µl/l) with lysozyme (0.5 mg/ml) and DNAse I (10 µg/ml) and further lysed using a French Press or sonicator (for smaller cell pellets <2–3 g). The lysate was clarified by centrifuging twice at 18 000 rpm at 4°C, 20 min each. The cleared lysate was divided in half. From one-half 70S ribosome was purified in the conventional method and the affinity-purification method was employed with the other half. In parallel, wild-type ribosome was also purified from the parent strain MG1655 in the conventional way for comparison.
For affinity purification, a HisTrapTMHP column (Ni2+–sepharose pre-packed, 5 ml, GE Healthcare) was connected to an ÄKTA prime chromatography system (GE Healthcare) equilibrated with the lysis buffer. After loading the lysate, the column was washed with 5 mM imidazole until A260 reached the baseline. The tetra-(His)6-tagged ribosomes were then eluted with 150 mM imidazole, pooled immediately and dialyzed 4 × for 10 min in 250 ml lysis buffer to remove the imidazole. Furthermore, the ribosomes were concentrated by centrifugation at 150 000 × g for 2 h at 4°C, resuspended in 1× polymix buffer containing 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate and 1 mM dithioerythritol (23 (link)) and shock-frozen in liquid nitrogen for storage or dissolved in the overlay buffer (20 mM Tris–HCl pH 7.6, 60 mM NH4Cl, 5.25 mM Mg acetate, 0.25 mM EDTA and 3 mM 2-mercaptoethanol) for sucrose gradient analysis. As a control, lysate from wild-type E. coli MG1655 was applied to the same column and was treated accordingly.
For purifying JE28 and MG1655 ribosomes in the conventional ultracentrifugation method (24 (link)), the cleared lysate was layered on top of equal volume of 30% w/v sucrose cushion made in a buffer containing 20 mM Tris–HCl pH 7.6, 500 mM NH4Cl, 10.5 mM Mg acetate, 0.5 mM EDTA, and 7 mM 2-mercaptoethanol and centrifuged at 100 000 × g for 16 h at 4°C. This step was repeated twice and in between the ribosome pellet was gently rinsed with the same buffer. Then the pellet was dissolved in 1× polymix buffer for storage or in the overlay buffer for sucrose gradient analysis as in case of the affinity-purified ones.
For affinity purification, a HisTrapTMHP column (Ni2+–sepharose pre-packed, 5 ml, GE Healthcare) was connected to an ÄKTA prime chromatography system (GE Healthcare) equilibrated with the lysis buffer. After loading the lysate, the column was washed with 5 mM imidazole until A260 reached the baseline. The tetra-(His)6-tagged ribosomes were then eluted with 150 mM imidazole, pooled immediately and dialyzed 4 × for 10 min in 250 ml lysis buffer to remove the imidazole. Furthermore, the ribosomes were concentrated by centrifugation at 150 000 × g for 2 h at 4°C, resuspended in 1× polymix buffer containing 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate and 1 mM dithioerythritol (23 (link)) and shock-frozen in liquid nitrogen for storage or dissolved in the overlay buffer (20 mM Tris–HCl pH 7.6, 60 mM NH4Cl, 5.25 mM Mg acetate, 0.25 mM EDTA and 3 mM 2-mercaptoethanol) for sucrose gradient analysis. As a control, lysate from wild-type E. coli MG1655 was applied to the same column and was treated accordingly.
For purifying JE28 and MG1655 ribosomes in the conventional ultracentrifugation method (24 (link)), the cleared lysate was layered on top of equal volume of 30% w/v sucrose cushion made in a buffer containing 20 mM Tris–HCl pH 7.6, 500 mM NH4Cl, 10.5 mM Mg acetate, 0.5 mM EDTA, and 7 mM 2-mercaptoethanol and centrifuged at 100 000 × g for 16 h at 4°C. This step was repeated twice and in between the ribosome pellet was gently rinsed with the same buffer. Then the pellet was dissolved in 1× polymix buffer for storage or in the overlay buffer for sucrose gradient analysis as in case of the affinity-purified ones.
