Chromatography system for protein purification

用于蛋白质纯化的色谱系统

• 批准号: 391024-2010
• 负责人: Hill, Bruce
• 金额: $ 4.54万
• 依托单位: Queen's University
• 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: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=443350
• 关键词: Chromatography; system; protein; purification

My research program is focused on the membrane protein complexes of the respiratory electron transfer chain, and the accessory proteins that assist in the assembly of these complexes. Our present focus is on the enzyme complexes of the cytochrome oxidase family. We are interested in aspects of the cytochrome oxidase mechanism that results in the consumption of oxygen and storage of the resulting free energy that can be used for other cellular work. There are accessory proteins that help to construct proteins such as the cytochrome oxidase complex. Cytochrome oxidase consists of up to 13 protein subunits and these subunits have associated with them two iron ions and three copper ions. The metal ions play a direct role in the reactions catalyzed by cytochrome oxidase. In such metalloproteins the properties of the metal ions are modulated by the protein matrix and our approach to understand this subtle interplay begins with the purified protein. We use the purified oxidase to look at aspects of its reaction with oxygen, reductants and how these reactions are modulated by the interactions with membrane lipids. We are also exploring how the metal centers of the oxidase are assembled, and the protein molecules that are involved in this process. Each of our projects on the oxidase directly, or on the accessory proteins involved in its assembly begins with the purification of the protein from its biological source. These purifications involve a series of controlled steps that take place over 1-2 days, or up to one week in time. Our work relies heavily on the preparation of these proteins in large amounts in a highly purified state. Such demands require careful and controlled conditions to achieve reproducible, highly purified products in high yield. This outcome is most readily achieved through use of an automated chromatography system that affords the user a high degree of control over the purification process. The equipment requested here will allow personnel in my group to obtain the highly purified proteins that are a prerequisite for their detailed studies. In addition the use of the requested equipment supplies valuable training experience for technicians, undergraduate and graduate students in the fields of biochemistry and biophysics.

我的研究项目主要集中在呼吸电子传递链的膜蛋白复合物，以及辅助这些复合物组装的辅助蛋白。我们目前的重点是细胞色素氧化酶家族的酶复合物。我们感兴趣的方面的细胞色素氧化酶的机制，导致在消耗氧气和储存所产生的自由能，可用于其他细胞的工作。还有一些辅助蛋白质帮助构建蛋白质，如细胞色素氧化酶复合物。细胞色素氧化酶由多达13个蛋白质亚基组成，这些亚基与两个铁离子和三个铜离子缔合。金属离子在细胞色素氧化酶催化的反应中起直接作用。在这样的金属蛋白中，金属离子的性质由蛋白质基质调节，我们理解这种微妙相互作用的方法始于纯化的蛋白质。我们使用纯化的氧化酶来研究它与氧、还原剂的反应，以及这些反应如何通过与膜脂质的相互作用来调节。我们还在探索氧化酶的金属中心是如何组装的，以及参与这一过程的蛋白质分子。我们的每一个直接研究氧化酶或参与其组装的辅助蛋白的项目都是从生物来源中纯化蛋白质开始的。这些净化涉及一系列受控步骤，这些步骤在1-2天或长达一周的时间内进行。我们的工作在很大程度上依赖于这些蛋白质的大量制备在高度纯化的状态。这些要求需要仔细和受控的条件以高产率获得可再现的高度纯化的产物。该结果最容易通过使用自动化色谱系统实现，该系统为用户提供对纯化过程的高度控制。这里要求的设备将使我小组的人员能够获得高度纯化的蛋白质，这是他们进行详细研究的先决条件。此外，使用所要求的设备为生物化学和生物物理学领域的技术人员、本科生和研究生提供了宝贵的培训经验。