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Open AccessArticle

Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation

Enabling Technologies Group, Sanford Research, Sioux Falls, SD 57104, USA
Proteomics Facility, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD 57105, USA
Author to whom correspondence should be addressed.
Cells 2020, 9(5), 1070;
Received: 6 April 2020 / Revised: 22 April 2020 / Accepted: 23 April 2020 / Published: 25 April 2020
(This article belongs to the Collection Feature Papers in Cell Nuclei: Function, Transport and Receptors)
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. View Full-Text
Keywords: proximity-labeling; BioID; TurboID; biotinylation; nuclear pore complex; lamin proximity-labeling; BioID; TurboID; biotinylation; nuclear pore complex; lamin
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May, D.G.; Scott, K.L.; Campos, A.R.; Roux, K.J. Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation. Cells 2020, 9, 1070.

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