ArticleBMC biotechnology2008
In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system.
Article in BMC biotechnology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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Who cites it
39 citing papers in PubMed.
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- Zika Virus-Like Particles Bearing a Covalent Dimer of Envelope Protein Protect Mice from Lethal Challenge.Journal of virology · 2020Article
- Mammalian Expression andACS omega · 2020Article
- Mutation of the second sialic acid-binding site of influenza A virus neuraminidase drives compensatory mutations in hemagglutinin.PLoS pathogens · 2020Article
- Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS.Frontiers in synaptic neuroscience · 2020Article
- The 2nd sialic acid-binding site of influenza A virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance.PLoS pathogens · 2019Article
- Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation.PloS one · 2019Article
- From Protein Features to Sensing Surfaces.Sensors (Basel, Switzerland) · 2018Review
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundDue 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.
resultsWe 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.
conclusionThe 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.
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