Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation

Abstract

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.

Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks

“…However, biotin deficiency in culture condition could lead to potential abnormalities of the cells 37 . Moreover, miniTurboID-tag expression is very low, or the tag is very unstable 35,38 .…”

In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish

“…Of note, BASU and miniTurbo were expressed at a lower level than TurboID despite equal amount of RNA injection. While we did not explore the underlying reasons for this, greater instability of miniTurbo has been reported previously and other biotin ligases have shown poor expression in cell cultures (May et al, 2020;Branon et al, 2018). Therefore, we chose TurboID for all subsequent experiments.…”

Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

“…Directed evolution has been used to isolate enzymes with increased activity, increased stability, smaller molecular weight, and that are split into inactive fragments that reconstitute activity when combined. Smaller Pup substrates have also been identified increase the number of labeled background proteins relative to BioID (May, Scott, Campos, & Roux, 2020), perhaps due to its robust enzymatic activity.…”

“…In addition to being useful in spatial proteomics (Branon et al, 2018), TurboID has also proven successful to discover new protein–protein interactions (Larochelle, Bergeron, Arcand, & Bachand, 2019; Mair, Xu, Branon, Ting, & Bergmann, 2019; Tachie‐Menson et al, 2020; Zhang et al, 2019). However, for some bait proteins, TurboID may increase the number of labeled background proteins relative to BioID (May, Scott, Campos, & Roux, 2020), perhaps due to its robust enzymatic activity.…”