title: Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase.
abstract: Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka 820-8502, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Miyao
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka 820-8502, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka 820-8502, Japan.
authors.forename: Arata
authors.initials: A
authors.lastname: Shiraishi
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka 820-8502, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kawakami
authors.affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka 820-8502, Japan; Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, Tobata, Kitakyushu 804-8550, Japan. Electronic address: sueda@bio.kyutech.ac.jp.
authors.forename: Shinji
authors.initials: S
authors.lastname: Sueda
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000235:genetics*; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000378:metabolism; D015374:Biosensing Techniques; D054889:Fatty Acid Synthase, Type II / Q000235:genetics; D006046:Gold / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D006046:Gold; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein
keywords: Antibody immobilization; Biotin ligase; Biotinylation; Immunosensor; Protein A