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In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system
BACKGROUND: Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373293/ https://www.ncbi.nlm.nih.gov/pubmed/18423015 http://dx.doi.org/10.1186/1472-6750-8-41 |
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author | Predonzani, Andrea Arnoldi, Francesca López-Requena, Alejandro Burrone, Oscar R |
author_facet | Predonzani, Andrea Arnoldi, Francesca López-Requena, Alejandro Burrone, Oscar R |
author_sort | Predonzani, Andrea |
collection | PubMed |
description | BACKGROUND: Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism. RESULTS: We developed a single bigenic plasmid that contains two independent transcriptional units for the co-expression of both the protein tagged with BAP and an engineered version of the BirA enzyme. Upstream of the cDNA encoding BirA, a signal secretion leader sequence was added to allow translocation of the enzyme to the secretory pathway. Three different recombinant antibodies in the scFv format, a membrane bound and secretory truncated IgE Fc fragment and a soluble version of the human IgE high affinity receptor were shown to be efficiently biotinylated and to maintain their binding properties in immunofluorescence microscopy, flow cytometry and ELISA assays. CONCLUSION: The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific in vivo biotinylation of target molecules tagged with a short acceptor peptide. These molecules could be easily obtained from supernatants or extracts of mammalian cells and used for a wide range of biological applications. |
format | Text |
id | pubmed-2373293 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-23732932008-05-07 In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system Predonzani, Andrea Arnoldi, Francesca López-Requena, Alejandro Burrone, Oscar R BMC Biotechnol Research Article BACKGROUND: Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism. RESULTS: We developed a single bigenic plasmid that contains two independent transcriptional units for the co-expression of both the protein tagged with BAP and an engineered version of the BirA enzyme. Upstream of the cDNA encoding BirA, a signal secretion leader sequence was added to allow translocation of the enzyme to the secretory pathway. Three different recombinant antibodies in the scFv format, a membrane bound and secretory truncated IgE Fc fragment and a soluble version of the human IgE high affinity receptor were shown to be efficiently biotinylated and to maintain their binding properties in immunofluorescence microscopy, flow cytometry and ELISA assays. CONCLUSION: The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific in vivo biotinylation of target molecules tagged with a short acceptor peptide. These molecules could be easily obtained from supernatants or extracts of mammalian cells and used for a wide range of biological applications. BioMed Central 2008-04-18 /pmc/articles/PMC2373293/ /pubmed/18423015 http://dx.doi.org/10.1186/1472-6750-8-41 Text en Copyright © 2008 Predonzani et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Predonzani, Andrea Arnoldi, Francesca López-Requena, Alejandro Burrone, Oscar R In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title | In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title_full | In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title_fullStr | In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title_full_unstemmed | In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title_short | In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
title_sort | in vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373293/ https://www.ncbi.nlm.nih.gov/pubmed/18423015 http://dx.doi.org/10.1186/1472-6750-8-41 |
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