Protein expression, purification, and structural characterization

这是一个核心设施的建议，以支持计划项目的应用。这个核心的主要作用 (Core B）是表达和纯化将研究麻醉剂结合的蛋白质靶标， 为项目提供结构性支持。本核心提案的目标1和2侧重于生产和 纯化真核生物钾通道;这些蛋白质包括麻醉敏感性和- 不敏感通道（后者作为阴性对照，例如用于光标记实验）。 靶蛋白包括电压门控钾通道Shaw 2和Kvl.2。目标3侧重于 真核麻醉靶的原核同源物的生产和纯化，包括钠 通道NaChBac.所有这些蛋白质都将在异源系统中表达，并提供给我们的实验室。 同事，无论是在纯化，洗涤剂溶解的形式或作为粗膜馏分。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， 使未来的高分辨率分析麻醉剂结合。我们还将结晶和确定结构 用于与模型蛋白如脱铁铁蛋白结合的麻醉剂。

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.