title: In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.
abstract: Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.
authors: AlbaNova University Centre, KTH - Royal Institute of Technology, Department of Protein Technology, Stockholm, Sweden.
authors: Sara
authors: S
authors: Kanje
authors: Sophia
authors: S
authors: Hober
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*
chemical_list: D000596:Amino Acids; D000906:Antibodies; D001710:Biotin
keywords: Antibody labeling; Biomolecular engineering; Protein G; Site-specific biotinylation; Unnatural amino acid