Protein and Biochemistry

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PROJECT SUMMARY (See Instructions): The Protein and Biochemistry Core will express and purify proteins for all members of the Program Project. In addition. Core B will perform quantitative analyses of protein-DNA interacfions and characterize the kinefic properties of DNA repair proteins using high-throughput fluorimetric assays. Specific Aim 1: (A) To opfimize the expression and purificafion of DNA repair proteins Core B will produce mg amounts of soluble proteins for Projects 1, 2, 3, and 4. We will express the proteins in different E. coli strains using protocols and methods that have proven successful in the past four years in our laboratory, such as autoinducfion. We will continue to use limited proteolysis in conjunction with bioinformatics to delineate funcfional domains and express smaller fragments, if the full-length protein construct fails to overexpress. (B) To optimize the solubility and stability of proteins and complexes to be used in crystallizafion experiments, by characterizing solvent effects on protein aggregafion properties using dynamic light scattering and analytical gel filtrafion. Crystallization trials using commercial kits will be set up using a robofic workstafion. Specific Aim 2: To perform rapid quantitative analyses of protein-DNA interacfions and steady-state enzyme kinetic properties of wild-type and mutant DNA repair enzymes generated in this Program Project, using high-throughput fluorimetric assays