Repurposing Proximity-Dependent Protein Labeling (BioID2) for Protein Interaction Mapping in E. coli.
Methods Mol Biol
; 2828: 87-106, 2024.
Article
em En
| MEDLINE
| ID: mdl-39147973
ABSTRACT
Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.
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Base de dados:
MEDLINE
Assunto principal:
Biotinilação
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Mapeamento de Interação de Proteínas
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Escherichia coli
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article