2.6. Purification of Recombinant MBP-MS2 Coat Protein

E. coli expressing MBP-MS2 was a gift from Yongsheng Shi (University of California, Irvine, CA, USA). Cells were grown at 37 °C until OD600 reached 0.4, and protein expression was induced with 0.5 mM isopropyl β-d-thiogalactoside (IPTG). After 3 h incubation at 37 °C, cells were pelleted, resuspended in lysis buffer (20 mM Tris HCl, pH 7.5, 200 mM NaCl, 0.5 mM PMSF), and lysed by sonication on ice 3 times at 30-second intervals. Lysates were cleared by centrifugation at ~7500 rcf for 10 min at 4 °C. The supernatant was added to washed amylose resin (NEB). Samples were incubated overnight at 4 °C with micrococcal nuclease and 5 mM CaCl₂. The amylose resin was washed 3 times with lysis buffer, and MBP-MS2 was eluted with 5 mM Na₂PO₄, pH 7. Eluted protein was added to washed heparin agarose (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 4 °C. The heparin agarose was washed once with 5 mM Na₂PO₄, pH 7 and protein was eluted with buffer D-10% glycerol (20 mM HEPES, 100 mM KCl, 1 mM MgCl₂, 0.2 mM EDTA) for 15 min at 4 °C. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories).