Essential Equipment for Membrane Protein Preparation and Purification

GPCRs，也被称为7-跨膜结构域受体，负责激素，嗅觉，视觉和神经元信号传导。自2007年以来，已经确定了至少12种不同gpcr的高分辨率x射线晶体结构，包括与刺激GS蛋白络合的ß2-肾上腺素能受体（ß2AR）的无活性和活性状态。我们正在申请必要的设备，以便我们能够维持我们的核磁共振表达和纯化gpcr的设施。关键部件包括一个台式离心机，用于提取含有gpcr的细胞颗粒，进一步从裂解液中纯化蛋白质，并浓缩样品用于核磁共振。第二个组件是层流罩，用于细胞培养，例如用于表达ß2-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工作：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，以及4)GPCR功能动力学。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。该设备将使我们能够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。

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.