Full-length human RIG-I was previously cloned into a pET SUMO expression vector (19 (link)). This construct contains an N-terminal 6-His tag, followed by a SUMO tag and the complete human RIG-I protein sequence. A serine residue was also added between the SUMO tag and RIG-I to ensure efficient tag cleavage.
In this study, we added the amino acid sequence MPCCPGCCGS following RIG-I residue 190. This sequence was added using Q5 mutagenesis (New England Biolabs) according to the manufacturer’s instructions. AzF was incorporated by amber stop codon suppression. Thus, the wild-type amino acid Glu494 was mutated to an amber stop codon (UAG) by the QuikChange Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s recommendations.
In this study, we added the amino acid sequence MPCCPGCCGS following RIG-I residue 190. This sequence was added using Q5 mutagenesis (New England Biolabs) according to the manufacturer’s instructions. AzF was incorporated by amber stop codon suppression. Thus, the wild-type amino acid Glu494 was mutated to an amber stop codon (UAG) by the QuikChange Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s recommendations.
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