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 have 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 property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs 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 for the specific detection of human IgG1 by a solid-phase immunoassay.
Sibler
A P
AP
Laboratoire d'immunotechnologie et de microbiologie moléculaires, UPRES 1329, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Kempf
E
E
Glacet
A
A
Orfanoudakis
G
G
Bourel
D
D
Weiss
E
E
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001710:Biotin; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000276:immunology*; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA; D014157:Transcription Factors
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein