Molecular Biology, Protein Expression and Structural Biology

Core B Abstract The molecular biology, protein expression and structural biology core is organized around the pooling of the resources and expertise of the project leaders in the program. The conduct of this core is based on a model that has been successfully implemented in the past 15 years and has been extremely productive. This centralized resource provides service and assistance in molecular biology, bacterial/yeast expression and in the preparation of stable mammalian cell lines over-expressing vitamin K-dependent proteins, factor V variants and factor VIII variants. The core provides the facilities, hardware and assistance for large-scale protein production runs with stable or transiently transfected mammalian cells in serum-free conditioned media. The core will provide facilities for large scale isolation of recombinant proteins, the appropriate chromatography strategies and will maintain the affinity columns necessary for this activity. The core will also provide some quality control services particularly the quantitation of g-carboxyglutamic acid content to verify the integrity of expressed vitamin K-dependent proteins. Assistance and facilities will also be provided for the large-scale isolation of coagulation proteins from plasma. Core B personnel will assist program members in implementing sparse matrix crystal screens and provide expertise for optimization of crystallization conditions with expanded screens. The core will perform remote synchrotron data collection and assist with solving x-ray structures. The core supports high performance computers equipped with necessary programs for processing diffraction data, model building by molecular replacement and refinement. The core will also provide support for mail-in collection of small angle x-ray scattering data and their analysis. Cryo-EM analysis will be coupled with single particle reconstruction to obtain structural information. Hydrogen/deuterium exchange studies will be performed by Core personnel at the HDX core facility at Penn. The core will also assist with atomic force microscopy measurements. The Director will be responsible for the oversight and efficient conduct of these activities and for ensuring that equitable service is provided to the projects