High capacity protein expression and production incubators

高容量蛋白质表达和生产培养箱

• 批准号: RTI-2023-00467
• 负责人: Christendat, Dinesh
• 金额: $ 5.75万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746519
• 关键词: capacity; protein; expression; production; incubators

My group uses structural biology, biochemistry and bioinformatics approaches to investigate how the shikimate pathway and closely related metabolic pathways drive functional diversity in plants and microbes. The shikimate pathway allows organisms to adapt to environmental conditions for their growth and development. The pathway produces chorismate, a precursor for aromatic compounds including isoflavonoids, phenylpropanoids, lignin, chlorogenic acids, hormones etc. These compounds have important roles in biotechnological applications. Phenylpropanoids, isoflavonoids, and chlorogenic acids have powerful antioxidant properties with attractive nutritional benefits. The shikimate pathway is absent in animals but is essential in plants and microbes which provides important enzyme targets for the development of antimicrobials and herbicides. Therefore, there is tremendous interest in studying proteins from this pathway. We are requesting funding to purchase two refrigerated stackable high-capacity incubator shakers, model Innova S44i, ref 120V. These stackable incubators shakers are being requested to replace our old and broken incubator shakers. Our research depends on refrigerated shakers to conduct protein expression analysis and recombinant protein production. Specifically, our NSERC-DG funded research focuses on protein structural biology and protein biochemistry which makes purified proteins the most valuable currency in our studies. Therefore, the ability to advance our research program directly depends on having reliable refrigerated incubator shakers. Protein expression analysis are almost exclusively conducted at lower temperatures, between 16 ?C and 18 ?C. We found that this temperature range slows down the protein translation machinery and thus protein production, which allows sufficient time for proteins to independently fold without forming inclusion bodies. We initially had two refrigerated incubator shakers, one was designated for protein expression analysis and the other for protein preparation and they have been used for more than 20 years by my research group and other plant and microbial research labs in the Department of Cell and Systems Biology. However, both incubators stopped working during the pandemic. Quite fortunately we were able to salvage parts from one incubator to repair the other. As a result, our research activity has been impacted because only one of our refrigerated incubator shakers is functioning and it is highly unreliable and urgently needed to be replaced. If we are unable to replace these refrigerated incubator shakers my NSERC-DG funded research program and HQPs training in my research lab will be further delayed since all aspect of my research depends on our ability to successfully obtain recombinant proteins.

我的团队使用结构生物学，生物化学和生物信息学方法来研究莽草酸途径和密切相关的代谢途径如何驱动植物和微生物的功能多样性。莽草酸途径允许生物体适应环境条件以促进其生长和发育。该途径产生分支酸，芳香族化合物的前体，包括甘草酸，苯丙素，木质素，绿原酸，激素等。这些化合物在生物技术应用中具有重要作用。苯丙素、异黄酮和绿原酸具有强大的抗氧化作用，并具有诱人的营养价值。莽草酸途径在动物中不存在，但在植物和微生物中是必不可少的，它为开发抗微生物剂和除草剂提供了重要的酶靶标。因此，人们对研究来自这一途径的蛋白质产生了极大的兴趣。 我们正在申请资金购买两个冷藏可堆叠的高容量培养箱振荡器，型号为Innova S44 i，参考电压为120 V。这些可堆叠的培养箱振动筛被要求取代我们的旧的和破碎的培养箱振动筛。我们的研究依赖于冷冻振荡器进行蛋白质表达分析和重组蛋白生产。具体来说，我们的NSERC-DG资助的研究重点是蛋白质结构生物学和蛋白质生物化学，这使得纯化蛋白质成为我们研究中最有价值的货币。因此，推进我们的研究计划的能力直接取决于拥有可靠的冷冻培养箱摇床。蛋白质表达分析几乎完全在较低的温度下进行，在16？C和18？C.我们发现，这个温度范围减慢了蛋白质翻译机制，从而减慢了蛋白质的生产，这使得蛋白质有足够的时间独立折叠而不形成包涵体。我们最初有两个冷藏培养箱摇床，一个用于蛋白质表达分析，另一个用于蛋白质制备，我的研究小组和细胞与系统生物学系的其他植物和微生物研究实验室已经使用了20多年。然而，这两个孵化器在大流行期间停止工作。幸运的是，我们能够从一个孵化器中抢救出零件来修理另一个。因此，我们的研究活动受到了影响，因为我们的冷冻培养箱振荡器只有一个在工作，而且非常不可靠，急需更换。如果我们无法更换这些冷藏培养箱振荡器，我的NSERC-DG资助的研究计划和HQP培训将进一步推迟，因为我的研究的各个方面都取决于我们成功获得重组蛋白的能力。