Adequate amounts of a variety of high quality, purified protein are common requirements of each of the projects of this proposal. The Protein Core will use its expertise in molecular biology, protein expression, and protein purification to provide recombinant proteins for use in each of the projects of this Proposal. We have successfully fulfilled this role during the current funding period. The Protein Core has two main functions: (1) Generate purified recombinant proteins as needed by each of the projects. Optimal methods for expression, purification and, if necessary, reconstitution will be developed depending on the experimental requirements for the particular protein. Emphasis will be placed on maintaining structure, activity and function as similar as possible to native proteins. Toward these ends, extensive biochemical and functional characterizations will be done. Because several of the proposed studies consider the role of post-translational modifications (for example, phosphorylation or prenylation), careful attention will be given to assure homogeneous preparations of properly modified proteins. In addition, we will generate and purify antibodies against whole proteins or peptides. (2) Provide molecular biology support, which will include construction of expression plasmids, site-directed mutagenesis, yeast 2-hybrid assays to test for interacting proteins, and isolation of cDNA clones for proteins identified or needed by the projects.
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