In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We hav e previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co -expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this p roperty to develop a novel filter assay for the rapid identification of rec ombinant Fab reacting with immunoglobulin. Starting with total RNA of two n ewly established murine hybridoma cell lines producing anti-human IgG1 anti bodies, we selected in a single experiment the bacterial clones that expres sed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated F abs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful f or the specific detection of human IgG1 by a solid-phase immunoassay. (C) 1 999 Elsevier Science B.V. All rights reserved.