title: Activation of circularly permutated β-lactamase tethered to antibody domains by specific small molecules.
abstract: Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 β-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.
authors: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo , 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Miki
authors: M
authors: Kojima
authors: Hiroto
authors: H
authors: Iwai
authors: Jinhua
authors: J
authors: Dong
authors: Shean-Lee
authors: SL
authors: Lim
authors: Shigekazu
authors: S
authors: Ito
authors: Koichi
authors: K
authors: Okumura
authors: Masaki
authors: M
authors: Ihara
authors: Hiroshi
authors: H
authors: Ueda
mesh_terms: D007093:Imidazoles / Q000737:chemistry / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D000073943:Neonicotinoids; D009574:Nitro Compounds / Q000737:chemistry / Q000276:immunology*; D001618:beta-Lactamases / Q000737:chemistry / Q000276:immunology*
chemical_list: D007093:Imidazoles; D007128:Immunoglobulin Fragments; D000073943:Neonicotinoids; D009574:Nitro Compounds; C055966:immunoglobulin Fv; C082359:imidacloprid; D001618:beta-Lactamases