Superdex 75 16 60 gl

Wild-type and mutant proteins of human NCL–RRM2/3 were purified by similar protocols. Optimal protein expression condition was obtained from E. coli strain BL21 (DE3). Cells were harvested and lysed by sonication in buffer containing 25 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 5% glycerol, 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride (PMSF). The supernatant was subjected to Ni²⁺-NTA column and subsequent eluate was further polished by gel filtration chromatography (Superdex-75 16/60 GL, GE Healthcare) and monoQ GL column (GE Healthcare). The final purified protein was kept in buffer containing 20 mM HEPES (pH 7.4) and 100 mM KCl. All the buffers used were prepared in DEPC-treated water.

Protein purification was performed in two consecutive steps. The purification of (His)6-ExoR initially began with affinity chromatography using an ÄKTA Prime apparatus (GE Healthcare) and Nickel-Sepharose columns (HisTrap HP 1 ml, GE Healthcare) and was continued by size-exclusion chromatography using an ÄKTA FPLC apparatus (GE Healthcare) and a gel filtration column (Superdex 75 16/60 GL, GE Healthcare). For details, see Supplementary Material.