Ni chelating gravity column

The RNA-binding domain of yeast Puf5p (residues 201–600) was subcloned into the pSMX vector with an N-terminal His₆-SUMO tag40 (link). Escherichia coli cells BL21 Star (DE3) carrying the Puf5p plasmid were grown in Terrific Broth media to OD₆₀₀=∼0.8 and then protein expression was induced with 0.4 mM isopropylthiogalactoside for 20 h at 18 °C. The cell pellet was resuspended in lysis buffer containing 20 mM Tris, pH 8.0; 0.5 M NaCl; 20 mM imidazole; 5% (v/v) glycerol and 0.1% (v/v) β-mercaptoethanol and sonicated on ice. The lysate was cleared by centrifugation and loaded onto a Ni-chelating gravity column (Thermo Scientific). His-SUMO-tagged Puf5p was eluted with a buffer containing 20 mM Tris, pH 8.0; 50 mM NaCl; 0.2 M imidazole and 1 mM DTT. Ulp1 protease was added to remove the His₆-SUMO tag, and the protein solution was loaded onto a Hi-Trap heparin column (GE Healthcare) and eluted with a gradient from 0 to 1 M NaCl in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT. The fractions containing Puf5p were pooled and concentrated by Amicon filters and loaded onto a Superdex 200 16/60 column equilibrated in 20 mM HEPES, pH 7.4; 0.15 M NaCl and 2 mM DTT. The Puf5p peak fractions were pooled and concentrated in column buffer for crystallization and RNA-binding assays.