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Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation
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 ligas...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290721/ https://www.ncbi.nlm.nih.gov/pubmed/32344865 http://dx.doi.org/10.3390/cells9051070 |
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author | May, Danielle G. Scott, Kelsey L. Campos, Alexandre R. Roux, Kyle J. |
author_facet | May, Danielle G. Scott, Kelsey L. Campos, Alexandre R. Roux, Kyle J. |
author_sort | May, Danielle G. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7290721 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-72907212020-06-17 Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation May, Danielle G. Scott, Kelsey L. Campos, Alexandre R. Roux, Kyle J. Cells Article 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. MDPI 2020-04-25 /pmc/articles/PMC7290721/ /pubmed/32344865 http://dx.doi.org/10.3390/cells9051070 Text en © 2020 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ). |
spellingShingle | Article May, Danielle G. Scott, Kelsey L. Campos, Alexandre R. Roux, Kyle J. Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title | Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title_full | Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title_fullStr | Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title_full_unstemmed | Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title_short | Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation |
title_sort | comparative application of bioid and turboid for protein-proximity biotinylation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290721/ https://www.ncbi.nlm.nih.gov/pubmed/32344865 http://dx.doi.org/10.3390/cells9051070 |
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