Layer-by-layer assembly of enzyme-loaded halloysite nanotubes for the fabrication of highly active coatings.

The development of sturdy enzyme-containing hydrophilic coatings is important for applications such as water purification or biological sensing. Here, we investigate the encapsulation of a model enzyme (beta-lactamase, BlaP) into aluminosilicate halloysite nanotubes (HNTs), and their subsequent use for the fabrication of enzymatic coatings by layer-by-layer (LbL) assembly. Highly stable suspensions of enzymatically-active halloysite nanotubes were obtained by alkaline treatment of HNTs, followed by enzyme adsorption into the lumen of the nanotubes and of poly(ethylene imine) (PEI) onto their outer surface. Bioactive thin films based on the LbL-assembly of these modified nanotubes with negatively-charged alginate provided coatings with a significantly higher enzymatic activity compared to films in which the enzyme is not incorporated in the nanotubes. The obtained results show that the encapsulation of an enzyme in halloysite nanotubes is a viable route towards stable bioactive coatings, which could be easily adapted to entrap other types of biomacromolecules with the aim of preparing thin films for air or effluent decontamination.

Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition.

Monoclonal antibodies are playing an increasing role in both human and animal health. Different strategies of protein and chemical engineering, including humanization techniques of non-human antibodies were applied successfully to optimize clinical performances of antibodies. Despite the emergence of techniques allowing the development of fully human antibodies such as transgenic Xeno-mice, antibody humanization remains a standard procedure for therapeutic antibodies. An important prerequisite for antibody humanization requires standardized numbering methods to define precisely complementary determining regions (CDR), frameworks and residues from the light and heavy chains that affect the binding affinity and/or specificity of the antibody-antigen interaction. The recently generated deep-sequencing data and the increasing number of solved three-dimensional structures of antibodies from human and non-human origins have led to the emergence of numerous databases. However, these different databases use different numbering conventions and CDR definitions. In addition, the large fluctuation of the variable chain lengths, especially in CDR3 of heavy chains (CDRH3), hardly complicates the comparison and analysis of antibody sequences and the identification of the antigen binding residues. This review compares and discusses the different numbering schemes and "CDR" definition that were established up to date. Furthermore, it summarizes concepts and strategies used for numbering residues of antibodies and CDR residues identification. Finally, it discusses the importance of specific sets of residues in the binding affinity and/or specificity of immunoglobulins.

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

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 ß-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 ß-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 ß-lactamase activity. This suggests that the nanobody and ß-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid ß-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.