Cht 2

The RIG-I gene was cloned in the protein expression vector pET28 SUMO and expressed as SUMO fusion proteins in Escherichia coli strain Rosetta (DE3) (Novagen). The protein was purified using a series of chromatography columns as published previously (3 (link)). The soluble lysate was fractionated through a HisTrap HP (GE Healthcare), followed by Ulp1 protease digestion to remove 6× His-SUMO tag, hydroxyapatite (CHT-II, Bio-Rad), and heparin Sepharose (GE Healthcare). The purified protein was dialyzed into 50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 5 mM DTT and 10% glycerol overnight at 4°C, frozen in liquid nitrogen and stored at –80°C.

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.

PA28αβ was purified as described (60 (link)). Briefly, recombinant PA28αβ was expressed in BL21-STAR E. coli and purified using strong anion exchange (HiTrapQ and MonoQ, GE Life Sciences), followed by hydroxyapatite (CHT-II, Bio-Rad), and finished with SEC (Superose 6 Increase, GE Life Sciences). Recombinant PA28γ was expressed in Rosetta E. coli and purified using Ni-NTA affinity resin (Qiagen) and followed with SEC (Superose 6 Increase, GE Life Sciences), as previously described (10 (link)). PA28γ-K188E was created using QuikChangeII Site Directed Mutagenesis Kit (Agilent) and purified using methods like PA28γ, as previously described (31 (link)). PA28γ-α, PA28γ-2XCys, PA28αβ2XLys, and PA28γΔIDR constructs were designed as G-Blocks with N-terminal 6XHis Tags (Integrated DNA Technologies) and cloned into pET11a plasmids. pET11a plasmids with successfully cloned G-blocks were transformed into BL21-STAR E. coli and purified using the PA28γ Ni-NTA purification methods. Concentrations were determined using a Bradford assay with bovine serum albumin as the reference protein.