AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions
Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2020-05-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/54983 |
| _version_ | 1811253065655779328 |
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| author | Kohki Kido Satoshi Yamanaka Shogo Nakano Kou Motani Souta Shinohara Akira Nozawa Hidetaka Kosako Sohei Ito Tatsuya Sawasaki |
| author_facet | Kohki Kido Satoshi Yamanaka Shogo Nakano Kou Motani Souta Shinohara Akira Nozawa Hidetaka Kosako Sohei Ito Tatsuya Sawasaki |
| author_sort | Kohki Kido |
| collection | DOAJ |
| description | Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions. |
| first_indexed | 2024-04-12T16:44:11Z |
| format | Article |
| id | doaj.art-58015ddcf4fb4ccc8dc53d30006056da |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:44:11Z |
| publishDate | 2020-05-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-58015ddcf4fb4ccc8dc53d30006056da2022-12-22T03:24:38ZengeLife Sciences Publications LtdeLife2050-084X2020-05-01910.7554/eLife.54983AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactionsKohki Kido0Satoshi Yamanaka1Shogo Nakano2Kou Motani3Souta Shinohara4Akira Nozawa5Hidetaka Kosako6Sohei Ito7Tatsuya Sawasaki8https://orcid.org/0000-0002-7952-0556Division of Cell-Free Life Science, Proteo-Science Center, Matsuyama, JapanDivision of Cell-Free Life Science, Proteo-Science Center, Matsuyama, JapanGraduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, JapanDivision of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, JapanDivision of Cell-Free Life Science, Proteo-Science Center, Matsuyama, JapanDivision of Cell-Free Life Science, Proteo-Science Center, Matsuyama, JapanDivision of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, JapanGraduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, JapanDivision of Cell-Free Life Science, Proteo-Science Center, Matsuyama, JapanProximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions.https://elifesciences.org/articles/54983BioIDcell-freeprotein-protein interactionenzymatic engineeringancestral sequence reconstructionproximity labeling |
| spellingShingle | Kohki Kido Satoshi Yamanaka Shogo Nakano Kou Motani Souta Shinohara Akira Nozawa Hidetaka Kosako Sohei Ito Tatsuya Sawasaki AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions eLife BioID cell-free protein-protein interaction enzymatic engineering ancestral sequence reconstruction proximity labeling |
| title | AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions |
| title_full | AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions |
| title_fullStr | AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions |
| title_full_unstemmed | AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions |
| title_short | AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions |
| title_sort | airid a novel proximity biotinylation enzyme for analysis of protein protein interactions |
| topic | BioID cell-free protein-protein interaction enzymatic engineering ancestral sequence reconstruction proximity labeling |
| url | https://elifesciences.org/articles/54983 |
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