Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions |
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| Authors: | Dr. Anna Perols Melina Arcos Famme Prof. Dr. Amelie Eriksson Karlström |
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| Affiliation: | KTH Royal School of Technology, School of Biotechnology, Division of Protein Technology, AlbaNova University Centre, Stockholm, Sweden |
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| 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. Z32BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z32BPA and azide‐Z32BPA for CuI‐catalyzed cycloaddition, as well as DBCO‐Z32BPA for Cu‐free strain‐promoted cycloaddition. The Z32BPA 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. |
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| Keywords: | antibodies chemoselective conjugation click chemistry photoaffinity labeling solid-phase synthesis |
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