Expression and purification of phage T7 ejection proteins for cryo-EM analysis
Nicholas A Swanson1, Ravi K Lokareddy1, Fenglin Li1
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
STAR Protocols
|November 26, 2021
Summary
Researchers purified and characterized phage T7 ejection proteins (gp14, gp15, and gp16). This work enables high-resolution cryo-electron microscopy studies of the DNA-ejectosome and related phage proteins.
Area of Science:
- Microbiology
- Structural Biology
- Virology
Background:
- Bacteriophages, particularly those in the Podoviridae family, package genomes into precursor capsids with internal ejection proteins.
- In phage T7, these proteins (gp14, gp15, and gp16) form a DNA-ejectosome in the host envelope for genome delivery.
Purpose of the Study:
- To describe the purification and characterization of recombinant T7 phage ejection proteins: gp14, gp15, and gp16.
- To provide a protocol adaptable for studying similar proteins in other bacteriophages.
Main Methods:
- Purification of recombinant gp14, gp15, and gp16.
- Characterization of purified ejection proteins.
- Application of the protocol for cryo-electron microscopy (cryo-EM) structure analysis.
Main Results:
- Successful purification and characterization of recombinant T7 gp14, gp15, and gp16.
- The developed protocol facilitated high-resolution cryo-EM structure determination of the T7 periplasmic tunnel.
Conclusions:
- The purification and characterization of T7 ejection proteins are crucial for understanding phage DNA delivery mechanisms.
- The described protocol is a valuable tool for structural studies of bacteriophage ejection systems, applicable to a broader range of phages.
