This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. New mass spectrometry approaches have revolutionized cell biology by identifying the majority of proteins expressed in a given cell type. The methodology has been extended to the dissection of protein-protein interactions through affinity tags and purification of protein complexes. Using state of the art mass spectrometry we will identify cellular proteins that interact with Salmonella secreted virulence factors (so called type three secreted effectors;TTS effector). Specifically we will use a new tandem affinity tag together with new methods of protein-protein cross-linking to dissect the inflammasome and how one TTS effector inhibits NFK? activation via interaction with the Nod signal transduction pathway. We have constructed vectors containing a tandem affinity tag that are suitable for expression in mammalian cells as well as in Salmonella. The tag encodes two 6x His tags separated by a biotinylation site allowing purification of the tagged proteins under fully denaturing conditions. We will cross-link proteins with low concentrations of formaldehyde and attempt to use this tag to purify protein complexes using capabilities in the NCRR Center.
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