2.12. Preparation of 15N-Labeled N

Recombinant N protein was expressed in BL21 (DE3) pLysS cells (Promega) by first, preculturing cells in the LB broth containing 50 μg/mL ampicillin overnight at 37 °C. The preculture was pelleted and was resuspended in 6 mL M9 medium (per one liter: 60 g divalent sodium phosphate, 30 g monovalent potassium phosphate, and 5 g NaCl) supplemented with Trace Elements (50 μg/mL EDTA, 8.3 μg/mL ferric chloride hexahydrate, 0.84 μg/mL zinc (II) chloride, 0.13 μg/mL copper (II) chloride dihydrate, 0.10 μg/mL cobalt (II) chloride hexahydrate, 0.10 μg/mL boric acid, and 0.016 μg/mL manganese (II) chloride hexahydrate), 1 mM MgSO₄, 0.3 mM CaCl₂, 1 μg/mL thiamin, 1 μg/mL biotin, and 1 mg/mL ¹⁵NH₄Cl. Then, 1 mL cells was added to 500 mL M9 medium containing 50 μg/mL ampicillin and incubated at 37 °C till OD600 reached 0.5. Next, IPTG was added to 5 mM, and the culture was incubated for 16 h at 23 °C. Cells were pelleted by centrifugation at 5000 RPM, 4 °C for 20 min in F9-6x1000 LEX rotor (Thermo Scientific) and resuspended in 50 mL E.coli Lysis Buffer (50 mM Tris-HCl pH8, 300 mM NaCl, 5% Glycerol, 5 mM imidazole, 23 ng/mL PMSF, 21 ng/mL TLCK, 21 ng/mL TPCK, 3.3 ng/mL leupeptin, and 3.3 ng/mL lima bean). Twenty milligrams lysozyme was added to cells, and after 20 min incubation at 4 °C, cells were lysed with a cell disruptor. The lysate was centrifuged for 45 min at 36,000 RPM, 4 °C in Ti45 rotor (Beckman, Indianapolis, IN, USA), and filtered through a 0.45 μm membrane.

The following purification steps were conducted at 4 °C. ¹⁵N-labeled N was bound to Profinity Ni-IMAC resin (BioRad, Hercules, CA, USA) by running the lysate through a gravity-flow column. The resin was washed with Wash Buffer (50 mM Tris-HCl pH 8, 500 mM NaCl, 10% glycerol, and 10 mM imidazole), and the protein was eluted with 10 mL Elution Buffer (50 mM Tris-HCl pH 8, 500 mM NaCl, 10% glycerol, 300 mM imidazole, and the protease inhibitors as listed in E.coli Lysis Buffer). The eluate was concentrated down by centrifugation in Amicon 10 MWCO column (Sigma). Aliquots of purified proteins were frozen with liquid nitrogen and were stored at −80 °C.