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Expression and purification of phage T7 ejection proteins for cryo-EM analysis

ABSTRACT: Summary Bacteriophages of the Podoviridae 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). Graphical abstract Highlights • Expression and purification of phage T7 ejection proteins in mg quantities• Reconstitution of gp15:gp16 DNA-ejectosome periplasmic tunnel in vitro• Pore formation assay reveals gp14 forms a constitutive pore• Vitrification of gp15:gp16 complex for cryo-EM single particle analysis Bacteriophages of the Podoviridae 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.

SUBMITTER: Swanson N

PROVIDER: S-EPMC8605104 | biostudies-literature |

REPOSITORIES: biostudies-literature

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