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.

The Use of a β-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, the presence of a flexible linker (e.g. Gly-Ser repetitions) to connect the carrier and the inserted protein was shown to dramatically increase the tolerance towards the insertion of large and structured proteins into a carrier one 30,12 .…”

“…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 .…”