期刊
ANALYTICAL BIOCHEMISTRY
卷 484, 期 -, 页码 113-121出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.05.010
关键词
Antibody immobilization; Immunosensor; Protein A; Biotinylation; Biotin ligase
资金
- Japan Science and Technology Agency
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. (C) 2015 Elsevier Inc. All rights reserved.
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