CORE--MEMBRANE PROTEIN EXPRESSION AND PURIFICATION

需要大量前列腺素的物理测量 内源性过氧化氢合酶(PGHS)和细胞色素C氧化酶(CcOX)是 分别是本计划项目的项目I-III和IV-V的核心。 这些研究包括光谱(拉曼、FTIR、EPR、IR)和 天然矿物的结晶学(X射线和中子衍射)测量 和突变体PGHS和CcOX。这五个项目加在一起将需要 60多个pGHS和25个CcOX突变体的大量表达和纯化 从1-100微克不等。这一核心将独自负责 这些蛋白质的生产和纯化，然后将是 要么提供给结晶核心，要么提供给各个项目 直接用于分析。这一核心的目标是： 特异性目标1-构建杆状病毒表达载体 前列腺素内源性过氧化氢合酶-1和-2同工酶， 在夜蛾细胞中表达这些蛋白，并对它们进行纯化，为 项目中概述的结晶、光谱和物理研究 1比3。我们还将从公羊精囊中提纯酶用于 使用本地PGHS-1的实验。 特异靶2-表达天然和突变型细胞色素C氧化酶 利用pYJ123H表达载体的衍生物在 球形鲁氏杆菌菌株JS100和YZ200的分离纯化 这些蛋白质的结晶和光谱学研究概述 在项目4和5中。 所涉及的大量蛋白质意味着这一点 这项工作需要几名技术人员的专心努力。我们觉得 在5年内生产超过85个纯化蛋白的物流可以 通过服务核心实现最高效、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.