Native Deglycosylation and Size Exclusion Chromatography of Viral Chemokine Binding Proteins for Structural Discovery

Structural discovery of viral chemokine binding proteins can provide valuable information on the binding domains and protein-protein interfaces (PPI) of these immunologically relevant proteins. Protein expression in mammalian cells produces high-quality protein compared to other expression methods; however, because structural discovery methods such as cryo-EM-based single particle analysis (SPA) and x-ray crystallography use methods which combine data from many individual proteins, these demand a highly monodispersed sample composed of protein with ordered structure. These techniques are often incompatible with flexible glycosyl groups commonly present on proteins produced by mammalian cells and require deglycosylation to enable observation of the conserved tertiary structure beneath these variable, flexible, glycans. Using the Myxoma viral protein M-T7 as a test case, we discuss considerations and preliminary bioinformatic analysis for approaching structural discovery using freely accessible sequence and structure databases to maximize success and guide experiments. We describe a simple deglycosylation optimization protocol utilizing Endo H followed by size exclusion chromatography (SEC) based purification to produce and validate protein suitable for structural discovery. Considerations such as protein concentration and volumes required for crystallography and negative stain electron microscopy are discussed as well as grid blotting techniques for negative stain experiments to validate protein quality.