<it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system
<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo </it>can be...
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| Format: | Article |
| Language: | English |
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BMC
2008-04-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/8/41 |
| _version_ | 1818155550194532352 |
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| author | López-Requena Alejandro Arnoldi Francesca Predonzani Andrea Burrone Oscar R |
| author_facet | López-Requena Alejandro Arnoldi Francesca Predonzani Andrea Burrone Oscar R |
| author_sort | López-Requena Alejandro |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 <it>in vivo </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific <it>in vivo </it>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.</p> |
| first_indexed | 2024-12-11T14:44:11Z |
| format | Article |
| id | doaj.art-e5d7eeb585244dada97b80b600aff1e6 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-11T14:44:11Z |
| publishDate | 2008-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-e5d7eeb585244dada97b80b600aff1e62022-12-22T01:01:46ZengBMCBMC Biotechnology1472-67502008-04-01814110.1186/1472-6750-8-41<it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector systemLópez-Requena AlejandroArnoldi FrancescaPredonzani AndreaBurrone Oscar R<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific <it>in vivo </it>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.</p>http://www.biomedcentral.com/1472-6750/8/41 |
| spellingShingle | López-Requena Alejandro Arnoldi Francesca Predonzani Andrea Burrone Oscar R <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system BMC Biotechnology |
| title | <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system |
| title_full | <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system |
| title_fullStr | <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system |
| title_full_unstemmed | <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system |
| title_short | <it>In vivo </it>site-specific biotinylation of proteins within the secretory pathway using a single vector system |
| title_sort | it in vivo it site specific biotinylation of proteins within the secretory pathway using a single vector system |
| url | http://www.biomedcentral.com/1472-6750/8/41 |
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