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

doi:10.1016/S0022-1759(99)00016-2

Journal of Immunological Methods

Volume 224, Issues 1–2, 22 April 1999, Pages 129-140

https://doi.org/10.1016/S0022-1759(99)00016-2 Get rights and content

Abstract

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.

Introduction

The biotin/avidin methodology is of practical importance in immunochemistry, especially when the antigen to be revealed is an immunoglobulin. Biotinylation of antibodies is generally accomplished by means of chemical reagents that cross-link biotin randomly to the free amino-groups of immunoglobulins. This process leads to the generation of heterogeneous preparations with frequent impairment of immuno-reactivity, particularly if critical lysine residues are modified.

Conjugation methods based on gene fusions are an attractive alternative to chemical procedures (Lindbach et al., 1993). Recent developments in biotechnology have shown that the biotin/streptavidin interaction can be exploited by appending overexpressed proteins with biotin `tags', mainly for purification purposes (Cronan, 1990; Schatz, 1993; Lesley and Groskreutz, 1997). In vivo biotinylation is usually obtained by fusing the biotin-acceptor coding sequence of proteins that act in vivo as biotin transporters to the gene encoding the protein of interest. Escherichia coli contains only one naturally biotinylated protein, the biotin carboxyl carrier protein (BCCP), which is specifically labelled by the biotin holoenzyme synthetase (BirA; Barker and Campbell, 1981). The BCCP has been extensively characterised (Li and Cronan, 1992; Athapilly and Hendrickson, 1995) and several reports have shown that various chimeric proteins containing the biotin-acceptor domain of BCCP are biotinylated by E. coli cells (Wang et al., 1996, Wang et al., 1997; Smith et al., 1998).

We recently constructed a plasmid vector (pHL4-bio) which permits the cloning and production of in vivo biotinylated binding fragments of antibodies (Fab) in E. coli cells (Weiss et al., 1994). The biotin-acceptor sequence of BCCP (aas 56–156; hereafter referred to as BCCP*) was linked to the Fd coding region of Fab and the chimeric Fab/BCCP* proteins were secreted in the bacterial culture medium. The approach successfully yielded bifunctional antibody fragments, but the extent of biotinylation was less than 15% of the total expressed fusions. It has been shown that quantitative biotin-labelling can be achieved in heterologous systems by overexpressing the BirA product in the transformed cells (Cronan, 1990; Reed and Cronan, 1991). We therefore decided to investigate whether plasmid-mediated co-synthesis of BirA in cells secreting Fab/BCCP* molecules could improve the labelling efficiency of the recombinant immunoglobulin fragments.

We report here the construction of vectors which allow the production of bacterially-expressed Fab/BCCP* proteins that are biotinylated to near completion. This possibility was used to develop a new method for the functional screening of recombinant anti-IgG Fab. The in vivo biotinylated anti-human IgG1 Fabs which were isolated by this method from two newly established murine hybridomas were found to be suitable for the specific detection of human IgG1 proteins.

Section snippets

Hybridoma cell lines

The hybridoma cell lines used in this study (clones 16-736 and 18-896) were obtained after immunisation of BALB/c mice with purified human polyclonal IgGs. The splenocytes were fused to myeloma P3x63Ag8.653 and the resulting hybridomas were screened for their capacity to agglutinate human red blood cells, previously sensitised with anti-rhesus-D (anti-Rh(D)) antibodies (Sallander et al., 1995; Glacet et al., unpublished observations). The antibodies secreted by the two selected hybridoma cell

Effect of BirA overexpression on Fab labelling

In a previous study we showed that 3–15% of the Fab/BCCP* fusion proteins expressed in the bacterial supernatant of pFab4/BCCP*-transformed E. coli cells were labelled with biotin (Weiss et al., 1994). The expression plasmid was previously referred to as pHL4-bio. The secreted Fd/BCCP* and L chain protein were found to be disulfide linked and the biotinylated Fab4/BCCP* complexes of the IgG1 antibody Mab4 to human tumor necrosis factor alpha (Orfanoudakis et al., 1994) were shown to retain both

Discussion

In this study, we have shown that an appropriate plasmid system permits the production of recombinant Fab fragments that are efficiently labelled with biotin in vivo. By overexpressing the BirA protein in cells secreting Fab/BCCP* molecules, we found that the large majority of recovered fusions were biotinylated. This result is in agreement with the findings of other groups who have recently reported that the wild-type levels of BirA in E. coli are insufficient to completely biotinylate an

Acknowledgements

We thank Prof. Jean-François Lefèvre for constant encouragement and support, and Dr. Annick Dejaegere and Prof. Yves Boulanger for critical reading of this manuscript. Also we are grateful to Georges Schwalbach for expert technical assistance and many of our collegues for useful suggestions and stimulating discussions. This work was supported by the Université Louis Pasteur of Strasbourg and the Ministère de la Recherche et de la Technologie (programme ACC-SV14).

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

Abstract

Introduction

Section snippets

Hybridoma cell lines

Effect of BirA overexpression on Fab labelling

Discussion

Acknowledgements

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J. Biol. Chem.

J. Biol. Chem.

Protein Exp. Purif.

Protein Exp. Purif.

Gene

Anal. Biochem.

Mol. Immunol.

J. Immunol. Methods

J. Biol. Chem.

Gene

J. Biol. Chem.

Gene

Gene

Anal. Biochem.

Anal. Biochem.