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Published on: September 10, 2020
A Novel Proximity Biotinylation Assay Based on the Self-Associating Split GFP1-10/11
Aditi S Kesari1, Uma K Aryal2, Douglas J LaCount1
1Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA.
Researchers developed a new proximity biotinylation method using split-green fluorescent protein (GFP) to improve the detection of protein-protein interactions. This enhanced technique offers more biologically relevant results compared to direct fusion methods.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Proximity biotinylation methods like BioID and BioID2 identify protein-protein interactions in living cells by tagging proteins with biotin ligases.
- These ligases activate biotin, which then covalently binds to nearby proteins, enabling their purification and identification via mass spectrometry.
Purpose of the Study:
- To develop a novel modification of proximity biotinylation by integrating a self-associating split-green fluorescent protein (GFP) system.
- To enhance the specificity and efficiency of identifying physiologically relevant protein-protein interactions.
Main Methods:
- Fused GFP11 to clathrin light chain (CLTB) and BioID2 to GFP1-10, exploiting their high-affinity interaction to recruit the biotin ligase.
- Utilized an inducible cell line expressing BioID2-GFP1-10 to facilitate the removal of non-specifically biotinylated proteins.
- Co-expressed the constructs and analyzed the resulting protein complexes and their localization.
Main Results:
- Co-expression of GFP11-CLTB and BioID2-GFP1-10 resulted in a fluorescent complex that co-localized with clathrin heavy chain.
- The modified proximity biotinylation system in the inducible cell line yielded a higher percentage of biologically relevant interactions compared to direct BioID2 fusion.
- Demonstrated the ability to monitor BioID bait protein expression and localization.
Conclusions:
- The novel split-GFP-based proximity biotinylation system enhances the identification of protein-protein interactions.
- This method improves specificity and allows for monitoring of bait protein behavior.
- Offers a valuable tool for studying molecular interactions in living cells.
