Application for 2 shaker incubators for heterologous expression of labeled proteins

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分子膜蛋白和几种可溶性蛋白的研究，这些蛋白是我们用细菌细胞培养出来的。培养大量的细胞培养物(25升)，然后收获以回收适当标记的蛋白质。虽然我们的一些蛋白质核磁共振样品可以从1-2升的细菌细胞培养物中培养出来，但我们希望表达一种G蛋白偶联受体(GPCR)蛋白质，该蛋白质需要在严格的生长条件下进行25升细胞培养才能制成单个核磁共振样品。我们的表达方案中有很大一部分涉及冷诱导和生长(4C)，因此我们要求为2个冷藏摇床培养箱(44升容量)提供资金。由于这将是我们工作的核心，如果没有内部摇床孵化器，我们根本无法前进。这一仪器还将使我们能够测试有关GPCRs的结构/功能分析和确定低分辨率结构的新想法和战略。目前，超过50%的药效团以GPCRs为靶标，但我们的整个蛋白质数据库只包含一个高分辨率结构。结构生物学在这一领域的重要性不可低估，我们相信我们的方法可以取得真正的进展。这些设备还将在今年夏天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索加大学正在进行的生物技术硕士研究生课程的一部分，并在多伦多大学的生物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蛋白质研究和高场强核磁共振方面的专业知识，为这一中心做出独特贡献。

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.