Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Predonzani, Andrea, Arnoldi, Francesca, López-Requena, Alejandro, Burrone, Oscar R
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2008
Materias:
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
_version_ 1782154364263071744
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
work_keys_str_mv AT predonzaniandrea invivositespecificbiotinylationofproteinswithinthesecretorypathwayusingasinglevectorsystem
AT arnoldifrancesca invivositespecificbiotinylationofproteinswithinthesecretorypathwayusingasinglevectorsystem
AT lopezrequenaalejandro invivositespecificbiotinylationofproteinswithinthesecretorypathwayusingasinglevectorsystem
AT burroneoscarr invivositespecificbiotinylationofproteinswithinthesecretorypathwayusingasinglevectorsystem