Protein Synthesis and Characterization

蛋白质是身体的主力，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。我们请求支持3个相关项目：1）研究蛋白质-蛋白质相互作用以获得疾病的机制见解; 2）工程化基于水凝胶的递送系统，允许控制释放或呈现多种蛋白质;（3）验证介导干细胞环境物理方面的蛋白质。 1)Emili小组已经报道了数千种蛋白质相互作用，包括酵母、小鼠、苍蝇和蠕虫的蛋白质相互作用图谱。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内的蛋白质相互作用。我们成功的关键是能够在3D中观察细胞内的蛋白质复合物，使我们能够确定蛋白质组装体的拓扑组织和亚细胞分布。 2)Shoichet实验室设计了一种用于基于亲和力的蛋白质递送的水凝胶系统，该系统可以在保持蛋白质生物活性的同时可预测地控制蛋白质释放。我们已经证明了该系统对几种蛋白质的控制释放的广泛适用性。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，这些特征控制了损伤后干细胞的激活。我们现在的目标是通过使用病毒方法原位操纵它们来验证介导这些相互作用的候选蛋白质，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3种集成仪器：1）用于高产量生产工程蛋白的大规模细菌培养系统; 2）能够分离大量细菌和蛋白质并浓缩病毒颗粒的离心机; 3）用于原位天然蛋白质组装体的3D纳米级成像的新显微镜功能。 该设备将在我们的实验室以及校园和行业内广泛使用。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职业做好准备，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.