CORE--EXPRESSION, CHARACTERIZATION AND CRYSTALLIZATION

表达，表征和结晶（ECC）核心将优化计划项目成员的蛋白质表达，以及为晶体学靶向蛋白质设计和工程定点或缺失突变体。此外，ECC核心将对蛋白质-DNA相互作用和复合物稳定性进行定量分析。我们将使用晶体学设备中的结晶机器人来建立酶与其DNA底物复合物的结晶试验。 目的1优化目的蛋白的表达。我们将在E.大肠杆菌细胞，通过改变细胞株和其他因素，如温度，或孵育介质。在E.大肠杆菌中表达的或无活性的蛋白质将在昆虫细胞中表达。我们将使用Gateway系统轻松地从细菌表达系统切换到真核表达系统。 目的2：设计和工程化晶体学靶蛋白的定点突变体或缺失突变体。我们将利用生物信息学核心A来选择在每个糖基化酶或重组酶中进行的突变，这些突变可能会影响其活性。 目标3：（A）使用高通量荧光测定法对酶活性进行快速定量分析，并优化用于结晶实验的蛋白质和复合物的溶解度和稳定性。为此，我们将使用有限的蛋白水解，预测无序区域和动态光散射的组合来描绘保留结合和切割DNA底物的能力的较小的域。我们将使用商业上可获得的结晶筛选和定制设计的不完全析因筛选来搜索重组酶和糖基化酶复合物的结晶条件。

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.