CORE--MEMBRANE PROTEIN EXPRESSION AND PURIFICATION

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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.