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) 分别为本计划项目的项目 I-III 和 IV-V 的核心。 这些研究包括光谱（拉曼、FTIR、EPR、IR）和 天然产物的晶体学（X 射线和中子衍射）测量 以及突变型 PGHS 和 CcOX。这五个项目总共需要 超过 60 种 PGHS 和 25 种 CcOX 突变体的大量表达和纯化 范围为1-100微克。该核心将全权负责 这些蛋白质的生产和纯化，然后将 提供给结晶核心或单个项目 直接进行分析。该核心的目标是： 具体目标1-构建杆状病毒载体以表达 天然和突变型前列腺素内过氧化物H2合酶-1和-2同工酶， 在斜纹夜蛾细胞中表达这些蛋白质，并纯化它们用于 项目中概述的结晶、光谱和物理研究 1-3.我们还将从公羊精囊中纯化酶，用于那些 使用天然 PGHS-1 的实验。 具体目标 2 - 表达天然和突变细胞色素 C 氧化酶 (CcOX) 使用 pYJ123H 表达质粒的衍生物的蛋白质 球形棒杆菌细菌菌株JS100和YZ200；并净化 这些蛋白质用于结晶和光谱研究概述 在项目 4 和 5 中。 所涉及的蛋白质的数量和数量意味着这 这项工作需要多名技术人员的共同努力。我们感觉 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.