Protocol for the expression, purification, and biochemical characterization of the innate immune sensor MDA5
Year: 2025
Type: article
Abstract: 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 • Procedures for the expression and purification of recombinant mouse MDA5 from E. coli • Procedures for quantifying the ATPase and RNA-binding activities of purified MDA5 • Visualization and biophysical characterization of MDA5-dsRNA filaments Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W4416441152