title: Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase.
abstract: Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-Tag (1-3) . Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-Tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, followed by analysis by flow cytometry and confocal microscopy. All ligands retained their original antigen-binding properties when fused to the SNAP-Tag. The combination of recombinant antibodies or protein ligands with the SNAP-Tag facilitates simple and efficient covalent modification with a broad range of substrates, thus providing a useful and advantageous alternative to existing coupling strategies.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrasse 6, 52074 Aachen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Kampmeier
authors.forename: Markus
authors.initials: M
authors.lastname: Ribbert
authors.forename: Thomas
authors.initials: T
authors.lastname: Nachreiner
authors.forename: Sofia
authors.initials: S
authors.lastname: Dembski
authors.forename: Florent
authors.initials: F
authors.lastname: Beaufils
authors.forename: Andreas
authors.initials: A
authors.lastname: Brecht
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D053285:CD30 Ligand / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D017730:Ki-1 Antigen / Q000276:immunology; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D008863:Microspheres; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D013194:Staining and Labeling; D013379:Substrate Specificity
chemical_list: D053285:CD30 Ligand; D005456:Fluorescent Dyes; D017730:Ki-1 Antigen; D008024:Ligands; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C505774:TNFSF8 protein, human; D001710:Biotin; D012822:Silicon Dioxide; D019853:O(6)-Methylguanine-DNA Methyltransferase