title: Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains.
abstract: Allosteric control of enzyme activity with exogenous substances has been hard to achieve, especially using antibody domains that potentially allow control by any antigens of choice. Here, in order to attain this goal, we developed a novel antibody variable region format introduced with circular permutations, called Clampbody. The two variable-region domains of the antibone Gla protein (BGP) antibody were each circularly permutated to have novel termini at the loops near their domain interface. Through their attachment to the N- and C-termini of a circularly permutated TEM-1 β-lactamase (cpBLA), we created a molecular switch that responds to the antigen peptide. The fusion protein specifically recognized the antigen, and in the presence of some detergent or denaturant, its catalytic activity was enhanced up to 4.7-fold in an antigen-dependent manner, due to increased resistance to these reagents. Hence, Clampbody will be a powerful tool for the allosteric regulation of enzyme and other protein activities and especially useful to design robust biosensors.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Iwai
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Kojima-Misaizu
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology , Yokohama, Kanagawa 226-8503, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology , Yokohama, Kanagawa 226-8503, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D015675:Osteocalcin / Q000276:immunology*
chemical_list: D000906:Antibodies; D008024:Ligands; D015675:Osteocalcin