Protein Synthesis and Characterization

蛋白质是人体的主力，了解它们的作用、调节和相互作用是理解所有生物过程的关键。我们请求为3个相关项目提供支持：1)研究蛋白质相互作用以获得疾病的机制洞察力；2)设计水凝胶递送系统，允许多种蛋白质的受控释放或呈现；(3)验证与干细胞环境物理方面有关的蛋白质。 1)埃米利小组已经报告了数千种蛋白质相互作用，包括酵母、老鼠、苍蝇和蠕虫的蛋白质相互作用图。我们现在的目标是记录正常和患病的原代人类细胞和组织中的蛋白质相互作用。我们成功的关键是能够在3D中观察细胞内的蛋白质复合体，使我们能够确定蛋白质组装的拓扑组织和亚细胞分布。 2)Shoichet实验室设计了一种基于亲和力的蛋白质输送水凝胶系统，该系统可以预测地控制蛋白质释放，同时保持蛋白质的生物活性。我们已经证明了该系统对几种蛋白质的控制释放具有广泛的适用性。我们现在的目标是重新设计我们的系统，以允许同时但独立地释放多种蛋白质。这些实验依赖于我们从细菌宿主中生产大量工程蛋白的能力。 3)吉尔伯特实验室确定了骨骼肌干细胞环境的物理特征，这些特征控制着损伤后干细胞的激活。我们现在的目标是验证介导这些相互作用的候选蛋白质，方法是使用病毒方法在原位操纵它们，使用Shoichet实验室设计的蛋白质刺激它们，并直接可视化它们的亚细胞分布。 我们提出了我们成功所必需的三种集成仪器：1)用于高产生产工程蛋白的大规模细菌培养系统；2)能够分离大量产生的细菌和蛋白质并浓缩病毒颗粒的离心机；3)新的显微镜功能，用于原位对天然蛋白质组装进行3D纳米成像。 该设备将在我们的实验室内以及整个校园和行业中广泛使用。本科生、研究生和博士后研究员将接受这种尖端设备的培训，为他们在工业、学术界和政府的高级研究生涯做好准备，最终使加拿大和知识经济受益。

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.