Protocol for the expression, purification, and biochemical characterization of the innate immune sensor MDA5

MDA5 is one of the primary eukaryotic innate immune sensors of viruses, recognizing long double-stranded RNA (dsRNA). Here, we present procedures for the recombinant expression and purification of murine MDA5 from E. coli. We describe the steps to purify MDA5 in high yields for downstream experiments and procedures to determine the ATPase activity and RNA-binding properties of purified MDA5. These approaches can be used to produce disease-associated mutants of MDA5, to uncover the biochemical mechanisms underpinning known disease phenotypes. For complete details on the use and execution of this protocol, please refer to Singh et al.¹.