Core D: Protein Production Core

核心 D：蛋白质生产核心

• 批准号: 7777115
• 负责人: KEITH W MILLER
• 金额: $ 35.64万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777115
• 关键词: Affinity Chromatography; Agonist; Antibody Affinity; Biochemical; Bioreactors; Brain; Cell Line; Core Facility; Development; Epitopes; Equilibrium; Gated Ion Channel; General anesthetic drugs; Goals; Human; Instruction; Laboratories; Lead; Ligands; Lipid Bilayers; Mammalian Cell; Minor; Mission; Mutation; Neurons; Pharmacology; Positioning Attribute; Production; Proteins; Research Personnel; Resources; Services; Site; Synaptic Receptors; Techniques; Terminology; Tetanus Helper Peptide; Time; base; design; design and construction; experience; member; milligram; mutant; programs; receptor; reconstitution; stable cell line

The Protein Production Core's mission is to produce heterologously expressed, purified and reconstituted human neuronal receptors in the large quantities required for photolabeling and sequencing. This is a challenging resource-intensive objective best handled by a central facility. During the four years of its existence, the Core has thoroughly developed several inducible mammalian cell lines capable of expressing a number of Cys-loop ligand-gated ion channel receptors bearing accessible purification epitopes. With such cell lines the successive steps of solubilization, purification and reconstitution have been optimized. Two receptors are now in production in such cell lines, and it is proposed to developed three more. Using its Bioreactor facility, the Core is now in a position to routinely fulfill its overall specific aim of supplying Projects 1 & 2 with the milligram quantities of well characterized, purified receptors functionally reconstituted into defined lipid bilayers that are required for photolabeling studies. The specific receptors to be produced in this way are all human neuronal members of the Cys-loop superfamily. The primary focus is on the inhibitory receptors, specifically the following GABAA receptors: synaptic receptors composed of subunits aip3y2L: extrasynaptic receptors composed of subunits a4p36, and aipS receptors to act as controls. The representative excitatory receptors in the Cys-loop superfamily will be the neuronal nicotinic a4p2 receptor and the 5-HT3A receptor. A subsidiary goal is to support the needs of investigators involved in electrophysiological studies (Raines and Forman). They will be able to achieve efficiencies by using the Core's resources both for the more straightfonward establishment of stable cell lines and for the development of mutants, although the primary responsibility remains with the investigator thereafter. This latter service will not detract from the Core's main labor-intensive goal of developing stable, inducible cell lines and over- expressing human neuronal receptors for photolabeling and biochemical studies. The Core will be situated in the Mallinckrodt Pharmacology Laboratory at MGH. RELEVANCE (See instructions):

蛋白质生产核心的使命是生产异源表达，纯化和重组 人神经元受体的大量光标记和测序所需的。这是一 具有挑战性的资源密集型目标最好由中央设施来处理。在其四年的 存在，核心已经彻底开发了几种诱导型哺乳动物细胞系能够表达 许多携带可接近纯化表位的Cys环配体门控离子通道受体。与这些 细胞系已经优化了溶解、纯化和重构的连续步骤。两 现在，在这些细胞系中已经开始生产受体，并建议再开发三种受体。使用其 生物反应器设施，核心现在能够定期实现其总体特定目标， 图1和2中所示的受体与毫克量的充分表征的、纯化的受体在功能上重构为 定义的脂质双层，这是光标记研究所需的。这种细胞中产生的特异性受体 所有的人都是Cys环超家族的神经元成员。主要关注的是抑制性 受体，特别是以下GABAA受体：由亚基aip 3 γ 2L组成的突触受体： 由亚基a4 p36组成的突触外受体和aipS受体作为对照。的 Cys环超家族中的代表性兴奋性受体是神经元烟碱α 4 β 2受体 和5-HT 3A受体。一个次要的目标是支持调查人员的需要， 电生理学研究（Raines和Forman）。他们将能够通过使用 核心的资源，既为更直接地建立稳定的细胞系，并为发展 突变体，虽然主要责任仍然是由研究人员之后。后一项服务将 不减损核心的主要劳动密集型目标，即开发稳定的、可诱导的细胞系， 表达人神经元受体用于光标记和生物化学研究。核心将位于 麻省总医院的马林克罗特药理学实验室 相关性（参见说明）：