Expression and purification of phage T7 ejection proteins for cryo-EM analysis
Nicholas Swanson, Ravi K. Lokareddy, Fenglin Li, Chun-Feng David Hou, Mikhail Pavlenok, Michael Niederweis, Gino Cingolani
Abstract
family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages. For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).
Topics & Concepts
PodoviridaeGenomePeriplasmic spaceBacteriophageRecombinant DNAComputational biologyCapsidPhagemidDNABiologyMolecular biologyChemistryBiophysicsGeneticsEscherichia coliGeneBacteriophages and microbial interactionsAdvanced Electron Microscopy Techniques and ApplicationsGenomics and Phylogenetic Studies