CORE--MEMBRANE PROTEIN EXPRESSION AND PURIFICATION

需要大量前列腺素的物理测量 内过氧化物H2合酶（PGHS）和细胞色素c氧化酶（CcOX）是 分别是本计划项目I-III和IV-V的核心。 这些研究包括光谱（拉曼，FTIR，EPR，IR）和 晶体学（X射线和中子衍射）测量 以及突变型PGHS和CcOX。这五个项目将需要 表达和纯化超过60个PGHS和25个CcOX突变体， 范围从1-100微克。这个核心将全权负责 这些蛋白质的生产和纯化， 供应给结晶核心，或个别项目 直接用于分析。该核心的目标是： 具体目的1-构建杆状病毒载体，用于表达 天然和突变的前列腺素内过氧化物H2合酶-1和-2同工酶， 在夜蛾细胞中表达这些蛋白质，并纯化它们， 结晶，光谱和物理研究概述项目 1-3.我们还将从公羊精囊中纯化酶， 使用天然PGHS-1的实验。 具体目标2-表达天然和突变型细胞色素C氧化酶（CcOX） 使用pYJ 123 H表达质粒的衍生物在大肠杆菌中表达蛋白质。 球形棒状杆菌菌株JS 100和YZ 200;以及纯化 这些蛋白质的结晶和光谱研究概述 在项目4和5中。 大量的蛋白质参与意味着， 这项工作需要几名技术人员的专门努力。我们觉得 在5年内生产超过85种纯化蛋白质的物流可以 通过服务核心进行最有效、最经济的管理。

Physical measurements requiring large quantities of prostaglandin endoperoxide H2 synthase (PGHS) and cytochrome c oxidase (CcOX) are central to projects I-III and IV-V, respectively, of this Program Project. These studied include spectroscopic (Raman, FTIR, EPR, IR) and crystallographic (X-ray and neutron diffraction) measurements of native and mutant PGHS and CcOX. Together, the five projects will require the expression and purification of over 60 PGHS and 25 CcOX mutants in amounts ranging from 1-100 micrograms. This core will have sole responsibility for the production and purification of these proteins, which will then be supplied either to the Crystallization core, or to the individual projects directly for analysis. The aims of this core are: Specific Aim 1- To construct baculovirus vectors for the expression of native and mutant prostaglandin endoperoxide H2 synthase-1 and -2 isozyme, to express these proteins in spodoptera cells, and to purify them for the crystallization, spectroscopic, and physical studied outlined in Projects 1-3. We will also purify enzyme from ram seminal vesicles for those experiments that employ the native PGHS-1. Specific Aim 2- To express native and mutant Cytochrome C Oxidase (CcOX) protein using derivatives of the pYJ123H expression plasmid in the Rodobacter sphaeroides bacterial strains JS100 and YZ200; and to purify these proteins for the crystallization and spectrographic studies outlined in Projects 4 and 5. The large number and quantities of the proteins involved means that this work will require the dedicated effort of several technicians. We feel that the logistics of producing over 85 purified protein over 5 years can be managed most efficiently and economically through a service core.