Enzymatic functionalization of a nanobody using protein insertion technology

Abstract

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.

Human Chitotriosidase: Catalytic Domain or Carbohydrate Binding Module, Who’s Leading HCHT’s Biological Function

“…The insertion site is located between residues Asp197 and Lys198 of the β-lactamase and the insertions of the gene fragments into the BlaP gene were performed as described in our previous studies 21, 30, 37, 38 . It is important to note that, in our previous work, we used a slightly longer ChBD CHIT1 that included the 72 C-terminal residues of HCHT (residues Pro395 to Asn466), however based on sequence alignments; we noticed that only the 49 C-terminal residues of ChBD CHIT1 were conserved.…”

Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein

“…In particular, the high specificity and affinity of nanobodies make them valuable tools for neutralizing antigens in cells[29]. Conventional antibodies have been tested as targeting drugs, but development has been limited due to poor stability and the high cost of production[28].…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…Periplasmic expression was also successfully exploited to produce nanobodies suitable for reacting with maleimide fluorophores [81], fusions between VHHs and nanoluciferase to use as bioluminescent immunoreagents [82], for recovering correct-folded nanobody-intein fusion proteins suitable for thioester reactions and finally for the fabrication of immunomicelles [83]. This expression route was also preferred for producing a hybrid molecule in which an anti-lysozyme nanobody was inserted into a solvent-exposed loop of β-lactamase [84]. Both moieties conserved their function after purification.…”