Noureddine Rhazi

Enzymatic functionalization of a nanobody using protein insertion technology

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays.

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase.

The specificity of the Streptomyces R61 penicillin‐sensitive D‐Ala‐D‐Ala peptidase has been re‐examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly‐L‐Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D‐Ala‐D‐Ala peptidases. The Gly‐L‐Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly‐L‐Xaa peptide or D‐Alanine are supplied as acceptors. Utilization of substrates in which the terminal ‐COO− group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibilty of the transpeptidation reaction.

The ponA Gene of Enterococcus faecalis JH2-2 Codes for a Low-Affinity Class A Penicillin-Binding Protein

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.

The Use of a β-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 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.

1H, 13C and 15N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins, BlaP197Q0 and BlaP216Q0, wherein a Pro-Gly dipeptide has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion.