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 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.


CONCLUSIONS
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.
Predonzani
Andrea
A
Molecular Immunology Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. predonza@icgeb.org
Arnoldi
Francesca
F
López-Requena
Alejandro
A
Burrone
Oscar R
OR
D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000235:genetics; D019677:Artificial Gene Fusion / Q000379:methods; D001692:Biological Transport / Q000235:genetics; D001710:Biotin / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D021382:Protein Sorting Signals / Q000235:genetics; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D014157:Transcription Factors
D000888:Antibodies, Anti-Idiotypic; D029968:Escherichia coli Proteins; C475664:FCER1A protein, human; D007141:Immunoglobulin Fc Fragments; D021382:Protein Sorting Signals; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D007073:Immunoglobulin E; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli