Champion pet sumo vector

The gene encoding HK97 small terminase (residues 1–161) was cloned in Champion pET SUMO vector (ThermoFisher)²⁴ . Mutants of small terminase were produced by site directed mutagenesis with the original plasmid as a template and a pair of oligos (Eurofins Genomics) containing mutated region.

The human RIG-I protein was expressed and purified as described previously (Ren et al., 2019 (link)). In brief, RIG-I was fused to an N-terminal 6xHis tag and a SUMO tag, followed by ULP1 digestion site in ChampionTM pET SUMO vector (ThermoFisher Scientific). This construct was overexpressed in E.coli Rosetta™ 2(DE3) Singles™ Competent Cells (Millipore Sigma). RIG-I expression was induced by IPTG (0.5 mM) when OD600 reached 0.6 and proceeded for 20–24 hours at 16 °C. The pellets were lysed in buffer (25 mM HEPES, pH 8.0, 300 mM NaCl, 10% Glycerol, 5 mM BME) supplemented with EDTA-free Protease Inhibitor Cocktail (Sigma), followed by nickel affinity chromatography using Ni-NTA Superflow beads (Qiagen). RIG-I was treated by ULP1 to remove the SUMO tag, followed by cation exchange and size exclusion chromatography, using a HiTrap Heparin HP column (GE Healthcare) and then a Superdex 200 Increase 10/300 GL column (GE Healthcare). RIG-I was pooled in a storage buffer (25 mM HEPES, pH 7.4, 200 mM NaCl, 5% Glycerol, 5 mM BME) for use in further experiments. RIG-I used in cryo-EM studies was pooled in buffer without glycerol.