title: Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.
abstract: Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.
authors.affiliation: Department of Bioengineering, University of Pennsylvania , 210 South 33rd Street, 240 Skirkanich Hall, Philadelphia, Pennsylvania 19104, United States.
authors.forename: James Z
authors.initials: JZ
authors.lastname: Hui
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsourkas
mesh_terms: D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity
chemical_list: D001798:Blood Proteins; D007074:Immunoglobulin G; C026080:plasma protein Z; D010649:Phenylalanine; C053764:benzoylphenylalanine