Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells
Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by prote...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-02-01
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| Series: | Life |
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| Online Access: | https://www.mdpi.com/2075-1729/12/2/300 |
| _version_ | 1797478636251512832 |
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| author | Arman Kulyyassov Yerlan Ramankulov Vasily Ogryzko |
| author_facet | Arman Kulyyassov Yerlan Ramankulov Vasily Ogryzko |
| author_sort | Arman Kulyyassov |
| collection | DOAJ |
| description | Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method. |
| first_indexed | 2024-03-09T21:34:31Z |
| format | Article |
| id | doaj.art-a28b39fdbd284c4b8ff5f2d327f99188 |
| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-09T21:34:31Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Life |
| spelling | doaj.art-a28b39fdbd284c4b8ff5f2d327f991882023-11-23T20:47:11ZengMDPI AGLife2075-17292022-02-0112230010.3390/life12020300Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in CellsArman Kulyyassov0Yerlan Ramankulov1Vasily Ogryzko2Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/5 Kurgalzhynskoye Road, Nur-Sultan 010000, KazakhstanRepublican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/5 Kurgalzhynskoye Road, Nur-Sultan 010000, KazakhstanUMR8126, Institut de Cancerologie Gustave Roussy, Universite Paris-Sud 11, CNRS, 94805 Villejuif, FranceProtein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.https://www.mdpi.com/2075-1729/12/2/300biotin acceptor peptide (BAP)protein–protein interactions (PPI)proximity utilizing biotinylation (PUB)site-specific biotinylation |
| spellingShingle | Arman Kulyyassov Yerlan Ramankulov Vasily Ogryzko Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells Life biotin acceptor peptide (BAP) protein–protein interactions (PPI) proximity utilizing biotinylation (PUB) site-specific biotinylation |
| title | Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells |
| title_full | Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells |
| title_fullStr | Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells |
| title_full_unstemmed | Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells |
| title_short | Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells |
| title_sort | generation of peptides for highly efficient proximity utilizing site specific biotinylation in cells |
| topic | biotin acceptor peptide (BAP) protein–protein interactions (PPI) proximity utilizing biotinylation (PUB) site-specific biotinylation |
| url | https://www.mdpi.com/2075-1729/12/2/300 |
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