Protein Purification and Biochemistry

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The overall objective of this Shared Resource Core is to enhance the capacity of the project investigators of this cooperative research center to determine the novel molecular mechanisms contributing to innate immune recognition of a variety of viral pathogens by contributing to the following center goals: 1) to investigate the role novel Pattern Recognition Receptors (PRR) as authentic receptors of viral nucleic acid which affect subsequent human innate immune responses to viral pathogens and (2) to capitalize on unique biochemical capabilities to quantify the ligand-binding functions of PRRs that are technically-challenging. The core will provide center investigators with purified innate immune signaling proteins as well as specialized assays of their biochemical activities that are relevant to signal transduction. The Core will capitalize on the expertise of Dr. Duncan in recombinant protein production and characterization, biochemical reconstitution of signaling pathways, and specific work in the field of innate immune signaling in order to provide this support. The core will generate these proteins and assay the ATP binding, ATP hydrolysis, and binding to virally derived nucleic acids of each protein in order to ensure biochemically active properly functioning protein is available for the studies proposed by center investigators. The inclusion of the Protein Purification and Biochemistry Core in this U19 application will provide a common source for recombinant signaling proteins, several of which are utilized in multiple projects. Because the production and characterization of these proteins is not routine and requires specific assay technologies for nucleotide binding and hydrolysis as well as nucleic acid binding, a single Shared resource Core will allow for efficient availability these reagents to multiple laboratories and the use of robust standardized methodologies in order to assess nucleic acid sensing functions by these proteins. Overall, this core will provide tools for each project to understand the mechanisms of viral nucleic acid detection and better assess the potential for these signaling systems to be used to develop novel anti-viral strategies based on host signaling pathways.