Core B: Protein Expression and Purification

SUMMARY: CORE B The Protein Expression and Purification Core will serve all Program Project investigators. Core B will express and purify proteins for Projects 1, 2, 3, and 4. The Core Director, Dr. Sylvie Doublié, has 25+ years’ experience in protein expression and purification. She will oversee the protein production aspect of the Core and directly supervise the staff in charge of protein expression and purification. Her lab was the first to engineer a construct of the polymerase domain of human DNA Polymerase θ that expresses well and is active. Dr. Rick Wood, the Co-Director, has extensive experience in the expression of human proteins in eukaryotic expression systems. His group was the first to express full-length, active human Pol θ. Core B will produce soluble proteins and their variants for Projects 1, 2, 3, and 4. Protein constructs will be expressed in different E. coli strains using protocols and methods, such as autoinduction, that have a track record of success. The Core will also optimize the solubility and stability of proteins and complexes to be used in biochemical and cellular assays, crystallization experiments and single-molecule studies, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Full length Pol θ and other human repair factor that cannot be expressed in E. coli will be expressed in human 293 cells. The Core is well equipped to express and purify proteins. Core B is also equipped for tissue culture work, having the necessary incubators, hoods, microscope, centrifuge, and storage facilities. 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 Purification Core will allow the different projects to achieve their research goals in a cost-effective manner and with unified quality control. Core B does not duplicate any University core facilities that would be available to our research team.