The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

• Published in: Journal of visualized experiments 2018-02 (132)
• Main Authors: Vandevenne, Marylène, Dondelinger, Mathieu, Yunus, Sami, Freischels, Astrid, Freischels, Régine, Crasson, Oscar, Rhazi, Noureddine, Bogaerts, Pierre, Galleni, Moreno, Filée, Patrice
• Format: Article
• Language: English
• Subjects: beta-Lactamases - metabolism; Biochemistry, biophysics & molecular biology; Biochimie, biophysique & biologie moléculaire; Bioengineering; Biological Assay - methods; Biosensing Techniques - methods; Humans; Life sciences; Sciences du vivant
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/55414

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP β-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the β-lactamase activity. This suggests that the nanobody and β-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid β-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.