Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation

• Published in: Cells (Basel, Switzerland) Vol. 9; no. 5; p. 1070
• Main Authors: May, Danielle G, Scott, Kelsey L, Campos, Alexandre R, Roux, Kyle J
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 25-04-2020; MDPI
• Subjects: A549 Cells; Animal models; Animals; BioID; Biotin; Biotinylation; Biotinylation - methods; Cell culture; Cell lines; Cloning; Culture media; Directed evolution; Doxycycline; Endoplasmic reticulum; Genetic Vectors - analysis; Genetic Vectors - genetics; Humans; Immunofluorescence; Immunoglobulins; Labeling; lamin; Ligases - metabolism; Medical research; nuclear pore complex; Protein interaction; Protein Interaction Domains and Motifs; Protein Interaction Mapping - methods; Protein purification; Proteins; Proteomics; Proteomics - methods; proximity-labeling; Toxicity; TurboID; United Kingdom > UK; United States > US; BioID; nuclear pore complex; biotinylation; lamin; proximity-labeling; TurboID
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9051070

BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.