Ni2 nitrilotriacetate

All the RIG-I constructs were sub-cloned into a modified pET28b vector with N-terminal SUMO fusion. Human RIG-I (1–925), Helicase-RD (232–925), and 2^nd CARD-Helicase-RD (97–925), CARDs (1–228) were overexpressed in E. coli strain Rosetta (DE3) (Novagen) as soluble proteins. The isolation of pure proteins involved three chromatographic steps: affinity column (Ni²⁺-nitrilotriacetate, Qiagen), hydroxyapatite column (CHT-II, Bio-Rad) and heparin sepharose column (GE Healthcare). An additional gel-filtration chromatography step (Hiload 16/26 Superdex 200, GE Healthcare) was added to purify RIG-I. Purified helicase-RD was further dialyzed overnight at 4°C into 50 mM HEPES pH 7.5, 50 mM NaCl, 5mM DTT, 10% glycerol, snap frozen in liquid nitrogen and stored at –80°C as reported previously (19 (link)). The RD (801–925) was expressed in E. coli BL21 Star (DE3) cells and the soluble fraction was purified to homogeneity using a Ni²⁺affinity column, cation exchange (HiTrap SP, GE Healthcare) and gel filtration chromatography.

Human RIG-I was expressed using pET28 SUMO vector in Escherichia coli strain Rosetta (DE3) (Novagen). The cell lysate soluble fraction was purified using a Ni²⁺-nitrilotriacetate (QIAGEN) column, followed by Ulp1 SUMO protease digestion to remove 6xHis-SUMO tag. It was further purified by hydroxyapatite (CHT-II, Bio-Rad) and heparin Sepharose column chromatography (GE Healthcare). Purified protein was dialyzed into 50 mM HEPES pH 7.5, 50 mM NaCl, 5mM MgCl₂, 5 mM DTT, and 10% glycerol overnight at 4°C, then snap frozen in liquid nitrogen and stored at −80°C (Jiang et al., 2011 (link)).
T7 RNA polymerase was expressed using pAR1219 vector in Escherichia coli strain BL21 (Novagen). Cell lysate was purified in three steps: SP-Sephadex (Sigma Aldrich), CM-Sephadex (Sigma Aldrich), and then DEAE-Sephacel (Sigma Aldrich). Finally, purified protein was dialyzed into Storage Buffer (20 mM sodium phosphate pH 7.7, 1 mM Na₃-EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 50% (v/v) glycerol) (Jia and Patel, 1997 (link)).