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Bacteriophages pp 117-136 | Cite as

Analyzing Phage–Host Protein–Protein Interactions Using Strep-tag® II Purifications

  • Jeroen De SmetEmail author
  • Hanne Hendrix
  • An Van den Bossche
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1898)

Abstract

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host–phage protein–protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host–phage protein–protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

Key words

Phage–host protein–protein interactions Affinity purifications Bacteriophage Pseudomonas aeruginosa Mass spectrometry 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jeroen De Smet
    • 1
    Email author
  • Hanne Hendrix
    • 2
  • An Van den Bossche
    • 2
  1. 1.Lab4FoodKU LeuvenCampus GeelBelgium
  2. 2.Laboratory of Gene TechnologyKU LeuvenLeuvenBelgium

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