title: Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.
abstract: Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy.
authors.affiliation: KTH Royal School of Technology, School of Biotechnology, Division of Protein Technology, AlbaNova University Centre, 10691, Stockholm, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Perols
authors.affiliation: KTH Royal School of Technology, School of Biotechnology, Division of Protein Technology, AlbaNova University Centre, 10691, Stockholm, Sweden.
authors.forename: Melina
authors.initials: M
authors.lastname: Arcos Famme
authors.affiliation: KTH Royal School of Technology, School of Biotechnology, Division of Protein Technology, AlbaNova University Centre, 10691, Stockholm, Sweden. amelie@biotech.kth.se.
authors.forename: Amelie
authors.identifier: http://orcid.org/0000-0002-0695-5188
authors.initials: A
authors.lastname: Eriksson Karlström
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002384:Catalysis; D045744:Cell Line, Tumor; D003300:Copper; D061565:Cycloaddition Reaction; D019793:Fluorescein / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D000068878:Trastuzumab / Q000737:chemistry
chemical_list: D000480:Alkynes; D001386:Azides; D001710:Biotin; D003300:Copper; D019793:Fluorescein; D007074:Immunoglobulin G; D010455:Peptides; D010649:Phenylalanine; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C053764:benzoylphenylalanine; C508053:ERBB2 protein, human
keywords: antibodies; chemoselective conjugation; click chemistry; photoaffinity labeling; solid-phase synthesis