Protein expression, purification, and structural characterization

这是一项关于核心设施的建议，以支持计划项目申请。这个核心的主要作用 (核心B)是表达和纯化将研究麻醉剂结合的蛋白质靶标，并 为该计划提供结构性支持。本核心提案的目标1和目标2侧重于生产和 真核钾通道的纯化；这些蛋白包括麻醉敏感型和- 不敏感的通道(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Kvl2。目标3侧重于 包括钠在内的真核麻醉药靶标原核同系物的生产和纯化 频道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 以净化的、洗涤剂增溶的形式或以粗膜部分的形式。结构支撑 将以结晶学专业知识的形式提供给该计划；这些专业知识将致力于 探索作为麻醉靶点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。我们还将明确和确定结构 用于与模型蛋白质结合的麻醉药，如载脂蛋白。

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.