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.

Creation of a Ligand-Dependent Enzyme by Fusing Circularly Permuted Antibody Variable Region Domains

“…However, in the presence of 1 μM BGP-C10, the specific activity significantly increased and the value was 4.66-fold higher than with no antigen peptide ( p < 0.01). Therefore, the antigen-dependent catalytic behavior of Cbody-cpBLA was clearly implemented, and the dependency is significantly higher than in the Fv-cpBLA that we reported previously …”

“…The pelleted cells were homogenized and centrifuged at 10 000 g for 60 min at 4 °C. The pellet precipitant was solubilized, and the fusion protein was purified with Talon resin (Clontech, Takara-bio) and refolded as previously described . The concentration of purified protein was measured with a Pierce BCA protein assay kit (Thermo scientific).…”

“…For example, the catalytic activity of two fusion proteins, each consisting of an antibody variable region and a truncated β-galactosidase fragment, was regulated through the reconstitution of the enzyme fragments induced by the antigen-Fv complex formation and applied to the sensitive homogeneous immunoassay of small haptens . Another example is a fusion protein consisting of a circularly permutated TEM-1 β-lactamase (cpBLA) and Fv . By use of cpBLA, which was originally reported as a part of single-molecule maltose-sensing switch that was fused with a circularly permutated maltose binding protein (cpMBP), , a fusion protein that could detect antigens was created by linking the native variable domains V H and V L to the N terminus and C terminus of cpBLA (Fv-cpBLA), respectively.…”

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

“…Therefore, it is important to mention that the size of the inserted protein has only little impact on the resulting chimeric protein. Indeed, it has been shown that large structured domains can be successfully inserted into β-lactamases, as long as their Nand C-terminal extremities are flexible or close to each other 13,14,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 . In the present study, the insertion site in BlaP is located far away from its active site and the inserted nanobody has relatively long and flexible extremities (not visible in the X-ray structure) that are located far away from the paratope.…”

“…Indeed, recent studies have shown that insertion of antibody variable fragments into the TEM1 β-lactamase results in a chimeric protein that remains able to bind with high affinity to its target antigen. Interestingly, the antigen binding was shown to induce allosteric regulation of TEM1 catalytic activity 11,12 . Furthermore, we showed in several studies that protein domain insertion into a permissive loop of the Bacillus licheniformis BlaP β-lactamase generates functional chimeric proteins that are well suited to monitor protein-ligand interactions 13,14 .…”