RIG-I Protein Purification Protocol

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.

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Schweibenz B.D., Solotchi M., Hanpude P., Devarkar S.C, & Patel S.S. (2023). RIG-I recognizes metabolite-capped RNAs as signaling ligands. Nucleic Acids Research, 51(15), 8102-8114.

Publication 2023

HisTrap HP (CHT-II, heparin Sepharose

Chromatography Digestion Escherichia coli Frozen Gene Glycerol Heparin sepharose Hepes Hydroxyapatite Mgcl2 Nacl Nitrogen Protein Rig i Strain Sumo protein Sumo proteins Ulp1 protease Vector

Corresponding Organization :

Other organizations : Rutgers, The State University of New Jersey, Johnson University

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Variable analysis

independent variables

Expression of RIG-I gene in pET28 SUMO vector
Expression of RIG-I gene in Escherichia coli strain Rosetta (DE3)

dependent variables

Purification of the RIG-I protein using a series of chromatography columns
Dialysis of the purified protein into a specific buffer
Freezing and storage of the purified protein

control variables

Concentration of buffer components (50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5 mM DTT, 10% glycerol)
Temperature (4°C for dialysis, -80°C for storage)

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