The Protein Expression and Cell Culture Core will provide expertise and support for all Program Project investigators. The Core will express and purify proteins for Projects 2, 3, and 4. In addition, Core B will also generate mammalian cell lines and provide cell culture services for all projects. The Core Director, Dr. Doubli, has extensive experience in protein expression and purification. She will continue to oversee the protein production aspect of the Core and directly supervise the staff in charge of protein expression, purification, optimization and preliminary crystallization screens. Dr. Sweasy, the Co-Director, has extensive experience in the construction of mammalian cell lines and in the characterization of DNA repair genes in tissue culture cells. Core B will produce mg amounts of soluble proteins for Projects 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 in our laboratory, such as autoinduction. The Core will also optimize the solubility and stability of proteins and complexes to be used in biochemical assays, crystallization experiments and single molecule studies, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Initial crystallization trials using commercial kits will be set up using a robotic workstation. Core B will also produce mammalian cell lines for all projects. The Core will screen the resulting cell lines for expression of the protein of interest. Mammalian proteins of interest will be expressed in HEK293 cells for biochemical experiments. The Core will also assist members of the Program with propagation of their cell lines and experiment design. The Core is well equipped to express and purify proteins. The technician in charge of protein production has worked in Core B for the last 8 years and is highly qualified. Core B is also well equipped for tissue culture work, having the necessary incubators, hoods, microscope, centrifuge, and storage facilities. A full-time technician with prior experience in mammalian cell culture techniques is in charge of the cell culture work. The services provided by Core B are essential for each Project and for the success of the Program Project as a whole. The Protein Expression and Cell Culture services are unique on the University of Vermont campus and do not duplicate any University core facilities already available to our research group.
Core B will play an integral role in providing purified proteins and mammalian cell culture lines to all Projects. We expect the results of the studies proposed within this Program Project to advance our understanding of how DNA repair protein variants contribute to cancer susceptibility.
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