Bacteriophages (phages) pose a constant threat to bacteria, driving the evolution of diverse anti-phage defense mechanisms. Among these defense strategies, recently identified anti-phage systems involve protein components of eukaryotic ubiquitination pathways. Structural characterization of these proteins is essential for elucidating their role in bacterial immunity and phage evasion strategies.
In this study, we focus on the structural and mechanistic characterization of key proteins from a bacterial anti-phage system. Using recombinant expression strategies and a multi-step purification protocol involving affinity and size-exclusion chromatography, we successfully purified all system proteins with high yield and purity. This accomplishment ensures the samples are well-suited for further structural analysis. We intend to use cryo-electron microscopy (cryo-EM) to gain a deeper understanding of their three-dimensional organization. This approach will enable high-resolution imaging of protein complexes, providing important insights into their structural organization and conformational dynamics. A deeper understanding of bacterial anti-phage systems could contribute to improving the stability of bacterial cultures, benefiting applications such as fermentation and probiotic production.