What is it about?
In this methods chapter, we describe a BioID2 toolkit for mapping protein-protein interactions inside living Escherichia coli cells. BioID2 adds a biotin tag to nearby proteins, which allows candidate interaction partners to be captured and analysed. We built plasmids that can attach BioID2 to either end of a protein of interest, and we demonstrate the workflow using CheA, a key protein in bacterial chemotaxis. In E. coli, CheA-BioID2 fusion proteins were expressed, produced biotin-labelled protein patterns that differed from BioID2 alone, and yielded captured protein samples suitable for follow-up mass spectrometry.
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Why is it important?
Protein interactions can be brief, condition-dependent, and hard to detect, so a live-cell labelling approach can help reveal candidates that might be missed by more targeted methods. Our toolkit adapts BioID2 for use in standard E. coli laboratory strains and provides a practical route from cloning through to capture of biotinylated proteins. The CheA example supports its use for studying bacterial chemotaxis and could help explore links between motility, DNA repair, and recombination systems.
Perspectives
From our perspective, the useful feature of this publication is that it brings the BioID2 workflow together as a practical protocol for E. coli, rather than only describing the concept. We show the key checks needed along the way: expression of the fusion proteins, in vivo biotinylation, comparison with BioID2 controls, and recovery of labelled proteins for further analysis. CheA was an interesting test case because the chemotaxis pathway is well studied, but still has open questions about protein partners, isoforms, and condition-dependent interactions.
Dr. Christian J Rudolph
Brunel University
Read the Original
This page is a summary of: Repurposing Proximity-Dependent Protein Labeling (BioID2) for Protein Interaction Mapping in E. coli, January 2024, Springer Science + Business Media,
DOI: 10.1007/978-1-0716-4023-4_9.
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