title: Optimal fusion of antibody binding domains resulted in higher affinity and wider specificity.
abstract: Antibody is a very important protein in biotechnological and biomedical fields because of its high affinity and specificity to various antigens. Due to the rise of human antibody therapeutics, its cost-effective purification is an urgent issue for bio-industry. In this study, we made novel fusion proteins PAxPG with a flexible (DDAKK)n linker between the two Ig binding domains derived from Staphylococcus protein A and Streptococcus protein G. The fusion proteins bound human and mouse IgGs and their fragments with up to 58-times higher affinity and wider specificity than the parental binding domains. Interestingly, the optimal linker for human Fab fragment was n = 4, which was close to the modeled distance between the termini of domains bound to heavy chain, implying increased avidity as a possible mechanism. For binding to Fc, the longest n=6 linker gave the highest affinity, implying longer interchain distance between the two binding sites. The novel fusion protein with optimized interdomain linker length will be a useful tool for the purification and detection of various IgGs including mouse IgG1 that binds only weakly to natural protein A.
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Kawasaki BIOLAB, Ushio Incorporated, 1-12 Minamiwatarida-cho, Kawasaki-ku, Kawasaki 210-0855, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ohashi
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan. Electronic address: ueda@res.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001665:Binding Sites / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D018583:Pliability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A
keywords: Affinity tag; Antibody binding protein; Avidity effect; Immunoassay; Linker engineering