Hiload superdex 200 size exclusion column

His₆-SUMO-tagged Acl4 and His₆-SUMO-tagged RpL4, encompassing residues 28–361 and 1–277, respectively, were coexpressed, as previously described10 (link). Filtered lysate was applied to a Ni-NTA column (Qiagen) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 500 mM NaCl and 5 mM β-ME and eluted with a linear imidazole gradient. Protein-containing fractions were pooled and cleaved with ULP1 and dialysed against a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME. Dialysed proteins were applied to a Ni-NTA column equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME and the unbound fraction was loaded onto a HiTrapQ HP (GE Healthcare) ion exchange column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM DTT and eluted with a linear salt gradient. Protein-containing fractions were pooled, concentrated and injected on a 16/60 HiLoad Superdex 200 size exclusion column (GE Healthcare) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM DTT. Protein-containing fractions were pooled, concentrated to ∼20 mg ml⁻¹ and used for crystallization.

Filtered lysate of His₆-SUMO tagged proteins was applied to a Ni-NTA column (Qiagen) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 500 mM NaCl and 5 mM β-ME and eluted with a linear imidazole gradient. Protein-containing fractions were pooled and cleaved with ubiquitin-like-specific protease 1 (ULP1) and dialysed against a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME (His₆-SUMO-RpL4^EXT was dialysed but not treated with ULP1). Dialysed proteins were applied to a Ni-NTA column equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME and the unbound fraction was loaded onto a HiTrapQ HP (GE Healthcare) ion exchange column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl, and 5 mM DTT and eluted with a linear salt gradient. Protein-containing fractions were pooled, concentrated and injected on a 16/60 HiLoad Superdex 200 size exclusion column (GE Healthcare) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM DTT. Protein-containing fractions were pooled, concentrated to ∼20 mg ml⁻¹ and flash frozen in liquid nitrogen for further use.

Rad5 was codon-optimized for bacterial expression and cloned into pET11a with an N-terminal 6xHis tag and a C-terminal Twin-Strep tag, resulting in plasmid pKW746. Rad5 was over-expressed in BL21 Star (DE3) cells by induction at an OD₆₀₀ of 0.6 with 1 mM IPTG for 16 hours at 16°C. Cells were lysed at 4°C using an EmulsiFlex (Avestin) in the presence of 1 mM PMSF, Complete, EDTA-free Protease Inhibitor Cocktail (Roche), and DNase. The crude extracts were clarified by ultracentrifugation. The proteins were purified using a Strep-Tactin XT resin (IBA) in 100 mM Tris pH 8.0, 150 mM KCl, 5% glycerol, and 1 mM DTT. Rad5 was eluted with 50 mM biotin and further purified using a HiLoad Superdex 200 size-exclusion column (GE Healthcare).