Application for 2 shaker incubators for heterologous expression of labeled proteins

2个摇床用于标记蛋白异源表达的应用

• 批准号: 359261-2008
• 负责人: Prosser, Scott
• 金额: $ 2.47万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2007
• 资助国家: 加拿大
• 起止时间: 2007-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=366494
• 关键词: Application; shaker; incubators; heterologous; expression

We propose to purchase two shaker incubators for purposes of heterologous expression of labeled proteins. My lab focuses on NMR-based studies of large membrane proteins and several soluble proteins, which we make using bacterial cell cultures. Large quantities of cell culture (25 litres) are grown and later harvested to recover appropriately labeled proteins. Although some of our protein NMR samples can be grown from 1-2 litre bacterial cell cultures, we wish to express a G protein-coupled receptor (GPCR) proteins which will require 25 litres of cell culture under stringent growing conditions, to make a single NMR sample. A large fraction of our expression protocols involve cold induction and growth (4C) and we therefore request funds for 2 refrigerated shaker incubators (a 44 litre capacity). As this will be central to our work, we simply cannot move forward without an in-house shaker incubator. This apparatus would also allow us to test new ideas and strategies regarding structure/function assays of GPCRs and the determination of low resolution structures. Over 50% of all pharmacophores currently target GPCRs yet our entire protein database consists of only one high resolution structure. The importance of structural biology in this field cannot be understated and we believe that our approach can make real inroads. The equipment will also be integrated into graduate learning based laboratory modules in the summer as part of the ongoing Master of Biotechnology graduate program at the University of Toronto Mississauga and used when needed by biology and chemistry colleagues at UTM. Through this investment, we also hope to establish better contacts with local chemical and biotechnology companies with overlapping research interests as part of the Western GTA Convergence Centre, for which we will be able to uniquely contribute through the capacity to heterologously express labeled proteins on a multi-litre scale, coupled with our expertise in biosynthetic labeling, GPCR protein research, and high-field NMR.

我们建议购买两个摇床培养箱用于标记蛋白的异源表达。我的实验室专注于基于核磁共振的大膜蛋白和几种可溶性蛋白的研究，这些蛋白是我们使用细菌细胞培养物制备的。培养大量细胞培养物（25 升）并随后收获以回收适当标记的蛋白质。虽然我们的一些蛋白质 NMR 样品可以从 1-2 升细菌细胞培养物中生长，但我们希望表达 G 蛋白偶联受体 (GPCR) 蛋白质，这需要在严格的生长条件下使用 25 升细胞培养物来制备单个 NMR 样品。我们的表达方案的很大一部分涉及冷诱导和生长 (4C)，因此我们请求资金购买 2 个冷藏摇床培养箱（容量为 44 升）。由于这将是我们工作的核心，因此如果没有内部摇床培养箱，我们根本无法前进。该装置还使我们能够测试有关 GPCR 结构/功能分析和低分辨率结构测定的新想法和策略。目前超过 50% 的药效团以 GPCR 为目标，但我们的整个蛋白质数据库仅包含一种高分辨率结构。结构生物学在这一领域的重要性不容低估，我们相信我们的方法可以取得真正的进展。该设备还将在夏季整合到基于研究生学习的实验室模块中，作为多伦多大学密西沙加分校正在进行的生物技术硕士研究生课程的一部分，并在 UTM 的生物学和化学同事需要时使用。通过这项投资，我们还希望与具有重叠研究兴趣的当地化学和生物技术公司建立更好的联系，作为西部 GTA 融合中心的一部分，我们将能够通过在多升规模上异源表达标记蛋白质的能力，加上我们在生物合成标记、GPCR 蛋白质研究和高场 NMR 方面的专业知识，为该中心做出独特的贡献。