1. Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.
- Author
-
Leskinen HL and Udvadia AJ
- Subjects
- Humans, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins genetics, Protein Binding, Mass Spectrometry methods, Protein Processing, Post-Translational, Endonucleases, Multifunctional Enzymes, Biotinylation, Protein Interaction Mapping methods, Transcription Factors metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase metabolism, DNA-(Apurinic or Apyrimidinic Site) Lyase chemistry
- Abstract
Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2025
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