The Expression, Characterization, and Crystallization (ECC) Core will optimize protein expression for Program Project members, as well as design and engineer site-directed or deletion mutants for proteins targeted for crystallography. In addition, the ECC Core will perform quantitative analyses of protein-DNA interactions and complex stability. We will use the crystallization robot in the Crystallographic Facility to set up crystallization trials of enzymes in complex with their DNA substrates.
Aim 1 To optimize the expression of proteins of interest. We will optimize protein expression in E. coli cells first, by varying the cell strains and other factors, such as temperature, or the incubation medium. The protein constructs that do not express well in E. coli or are not active will be expressed in insect cells. We will use the Gateway system to easily switch from the bacterial to the eukaryotic expression system.
Aim 2 : To design and engineer site directed or deletion mutants of the proteins targeted for crystallography. We will make use of Bioinformatics Core A to choose the mutation to make in each glycosylase or recombinase that would potentially affect its activity.
Aim 3 : (A) To perform rapid quantitative analyses of enzyme activity using high-throughput fluorimetric assays and to optimize the solubility and stability of proteins and complexes to be used in crystallization experiments. To this end, we will use a combination of limited proteolysis, prediction of disordered regions and dynamic light scattering to delineate smaller domains that retain the ability to bind and cleave DNA substrates. We will use both commercially available crystallization screens and custom-designed incomplete factorial screens to search for crystallization conditions for the recombinase and glycosylase complexes.
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