Protein expression, purification, and structural characterization

蛋白质表达、纯化和结构表征

• 批准号: 8902164
• 负责人: Patrick J Loll
• 金额: $ 21.12万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8902164
• 关键词: Anesthetics; Apoferritin; Architecture; Arts; Baculoviruses; Binding; Biochemical; Breathing; Carrier Proteins; Cells; Charge; Chimera organism; Chromatography; Complex; Core Facility; Crystallization; Cysteine; Cytoplasmic Tail; Detergents; Deuterium; Electrons; Engineering; Environment; Equipment; Future; General anesthetic drugs; Goals; Homologous Gene; Human; Insecta; Integral Membrane Protein; Interferometry; Ion Channel; Ion Channel Protein; Isotope Labeling; Label; Lysine; Measures; Membrane; Membrane Proteins; Metals; Modeling; Neutrons; Nuclear; Potassium Channel; Preparation; Production; Protein Truncation; Proteins; Quality Control; Reagent; Recombinant Proteins; Research; Resolution; Role; Saccharomyces cerevisiae; Site; Sodium Channel; Structure; System; Technology; Transmembrane Domain; Voltage-Gated Potassium Channel; Work; design; innovation; macromolecule; milligram; mutant; novel; overexpression; programs; protein expression; research study; structural biology; three dimensional structure

This is a proposal for a core facility to support a Program Project application. The main roles of this core (Core B) are to express and purify the protein targets for which anesthetic binding will be studied, and to provide structural support to the program. Aims 1 and 2 of this core proposal focus on the production and purification of eukaryotic potassium channels; these proteins include both anesthetic-sensitive and - insensitive channels (with the latter acting as negative controls, for example for photolabeling experiments). Target proteins include the voltage-gated potassium channels Shaw2 and Kvl.2. Aim 3 focuses on the production and purification of prokaryotic homologs of eukaryotic anesthetic targets, including the sodium channel NaChBac. All of these proteins will be expressed in heterologous systems and supplied to our colleagues, either in purified, detergent-solubilized form or as crude membrane fractions. Structural support will be supplied to the program in the form of crystallographic expertise; this expertise will be devoted to exploring crystallization conditions for the membrane proteins being studied as anesthetic targets, so as to enable future high-resolution analysis of anesthetic binding. We will also crystallize and determine structures for anesthetics bound to model proteins such as apoferritin.

这是支持计划项目申请的核心设施提案。该核心的主要作用 （核心 B）是表达和纯化将研究麻醉剂结合的蛋白质靶标，并 为该计划提供结构支持。该核心提案的目标 1 和 2 重点关注生产和 真核钾通道的纯化；这些蛋白质包括麻醉敏感蛋白和 - 不敏感通道（后者充当阴性对照，例如用于光标记实验）。 靶蛋白包括电压门控钾通道 Shaw2 和 Kvl.2。目标 3 侧重于 真核麻醉靶标的原核同系物的生产和纯化，包括钠 NaChBac 通道。所有这些蛋白质都将在异源系统中表达并提供给我们 同事们，无论是纯化的、洗涤剂溶解的形式还是粗膜组分。结构支撑 将以晶体学专业知识的形式提供给该计划；这种专业知识将致力于 探索作为麻醉靶点研究的膜蛋白的结晶条件，以便 使未来能够对麻醉剂结合进行高分辨率分析。我们还将结晶并确定结构 用于与脱铁铁蛋白等模型蛋白结合的麻醉剂。