Protein Synthesis and Characterization

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Proteins are the workhorses of the body and understanding their role, regulation, and interactions is key to understanding all biological processes. We request support for 3 related projects: 1) Studying protein-protein interactions to gain mechanistic insights of disease; 2) Engineering hydrogel-based delivery systems that allow controlled release or presentation of multiple proteins; (3) Validating proteins that mediate the physical aspects of the stem cell environment. 1) The Emili group has reported thousands of protein interactions, including protein interaction maps for yeast, mice, flies, and worms. We now aim to document protein interactions inside normal and sick primary human cells and tissues. Critical to our success is the ability to observe protein complexes within cells in 3D, allowing us to determine topological organization and subcellular distributions of protein assemblies. 2) The Shoichet lab has engineered a hydrogel system for affinity-based protein delivery that can predictably control protein release while maintaining protein bioactivity. We have demonstrated broad applicability of this system to the controlled release of several proteins. We now aim to re-engineer our system to allow simultaneous yet independent release of multiple proteins. These experiments depend on our ability to produce large amounts of engineered proteins from bacterial hosts. 3) The Gilbert lab identified physical features of the skeletal muscle stem cell environment that control stem cell activation after injury. We now aim to validate candidate proteins that mediate these interactions by manipulating them in situ using a viral approach, stimulating them using proteins engineered by the Shoichet lab, and directly visualizing their subcellular distribution. We propose 3 integrated instruments necessary for our success: 1) a large-scale bacterial culture system for high-yield production of engineered proteins; 2) a centrifuge capable of isolating the large amounts of bacteria and protein produced and to concentrate viral particles; 3) new microscope capabilities for 3D nanoscale imaging of native protein assemblies in situ. This equipment will be widely accessible within our labs and across campus and industry. Undergraduates, graduate students, and post-doctoral fellows will be trained on this cutting edge equipment, preparing them for advanced research careers in industry, academia and government, ultimately benefitting Canada and the knowledge-based economy.