title: Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain.
abstract: Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.
authors.affiliation: Department of Protein Technology, KTH - Royal Institute of Technology , SE-10691, Stockholm, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Kanje
authors.affiliation: Department of Protein Technology, KTH - Royal Institute of Technology , SE-10691, Stockholm, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: von Witting
authors.affiliation: HERM, Department of Medicine Huddinge, Karolinska Institute , SE-14157, Stockholm, Sweden.
authors.forename: Samuel C C
authors.initials: SC
authors.lastname: Chiang
authors.affiliation: HERM, Department of Medicine Huddinge, Karolinska Institute , SE-14157, Stockholm, Sweden.
authors.forename: Yenan T
authors.initials: YT
authors.lastname: Bryceson
authors.affiliation: Department of Protein Technology, KTH - Royal Institute of Technology , SE-10691, Stockholm, Sweden.
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D055668:Photochemical Processes; D000072757:Protein Conformation, beta-Strand; D000072417:Protein Domains; D013194:Staining and Labeling
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G