Essential Equipment for Membrane Protein Preparation and Purification

膜蛋白制备和纯化的必备设备

• 批准号: 440283-2013
• 负责人: Prosser, RobertScott
• 金额: $ 1.52万
• 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: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=507062
• 关键词: Essential; Equipment; Membrane; Protein; Preparation

GPCRs, also known as 7-transmembrane domain receptors, are responsible for hormonal, olfactory, visual, and neuronal signaling. Since 2007, the high-resolution X-ray crystal structures of at least a dozen distinct GPCRs have been determined, including the inactive and active states of the ß2-adrenergic receptor (ß2AR), complexed with the stimulatory GS protein. We are applying for essential apparatus so that we can maintain our facility for expression and purification of GPCRs for NMR. The key components include a bench-top centrifuge which is used extract cell pellets containing GPCRs, further purify the protein from lysate, and concentrate samples for NMR. The second component is a laminar flow hood which is used to work with cell cultures, such as insect cells which are used to express ß2-adrenergic receptor. The facility will also allow us to continue our ongoing work on 1) protein folding, 2) protein misfolding and amyloidosis, 3) enzyme functional dynamics, and 4) GPCR functional dynamics. In these studies we make use of either E coli or insect cells to express the proteins of interest. This equipment will allow us to maintain a leading role in fluorine NMR studies of complex proteins and macromolecules.

GPCRs，也被称为7-跨膜区受体，负责激素、嗅觉、视觉和神经元信号传递。自2007年以来，至少有十几个不同的GPCR的高分辨X射线晶体结构被确定，包括与刺激性GS蛋白络合的B_2-肾上腺素能受体(？2AR)的非活性和活性状态。我们正在申请必要的仪器，这样我们就可以维持我们的设施，表达和纯化核磁共振GPCRs。关键部件包括一台台式离心机，用于提取含有GPCRs的细胞球，进一步从裂解液中提纯蛋白质，并浓缩样品用于核磁共振。第二个部件是层流罩，用于处理细胞培养物，例如用于表达β2-肾上腺素能受体的昆虫细胞。该设施还将使我们能够继续我们正在进行的1)蛋白质折叠、2)蛋白质错误折叠和淀粉样变性、3)酶功能动力学和4)GPCR功能动力学的工作。在这些研究中，我们利用大肠杆菌或昆虫细胞来表达感兴趣的蛋白质。该设备将使我们在复杂蛋白质和大分子的氟核磁共振研究中保持领先地位。