The Eph receptors represent the largest family of receptor tyrosine kinases identified to date, with fourteen structurally similar Eph receptors now identified in the human genome. Eph receptors and ephrins are both anchored to the cell surface and play a key role in cell-cell communication, regulating such processes as tissue patterning, axonal guidance, and synaptic plasticity, angiogenesis and tumorigenesis. Both the Eph receptors and the ephrins can be divided into two classes based on sequence conservation. There are nine human EphA receptors and five ephrin-A ligands in the A class and there are five human Eph receptors and three ephrin-B ligands in the B class. The ephrins of the A class are GPI-anchored while the ephrins of the B class function as transmembrane proteins that contain conserved carboxy-terminal cytoplasmic domains. Interestingly, there is high binding promiscuity in the interactions between Eph receptors and ephrins of the same class, and several Eph receptors/ephrins can also mediate interactions between classes. The purpose of Core A is to provide protein expression and purification services to support the crystallographic, NMR, biochemical, and cell culture studies of the program project. The Eph Protein Production Core will generate purified Eph receptor and ephrin protein domains for Project 1-3. The Core will use both baculovirus and bacterial expression systems to produce protein domains that will be purified for crystallization trials, NMR experiments, biochemical, cell culture and in vivo studies. Filtration and column-based puriflcation methodologies will be used to generate purified proteins and Eph receptor/ephrin complexes. Size-exclusion chromatography, dynamic light scattering and mass spectrometry techniques will be used to assess complex formation and homogeneity and monodispersity of the purified proteins. Furthermore, the Core will design, generate and store wild-type and mutant Eph receptor and ephrin protein constructs. Relevant data from protein production runs will be stored on a SharePoint Server to enable data sharing amongst project participants
Significant expertise and effort are required to express and purify Eph receptors and ephrin protein domains for structural and biochemical studies to define the structural features of Eph receptor-ephrin binding specificity and their importance for tumor growth. Facilitating these studies through a centralized protein production core facility will also help the development of chemical compounds and peptides that will serve as potential cancer therapeutic starting points.
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