title: 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.
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: O
authors: O
authors: Crasson
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: N
authors: N
authors: Rhazi
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: O
authors: O
authors: Jacquin
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: A
authors: A
authors: Freichels
authors: Chimie des Macromolécules et des Matériaux Organiques (CERM), Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: C
authors: C
authors: Jérôme
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: N
authors: N
authors: Ruth
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
authors: M
authors: M
authors: Galleni
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium CER Groupe, Rue de la Science, n°8, Aye B6900, Belgium.
authors: P
authors: P
authors: Filée
authors: Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium mvandevenne@ulg.ac.be.
authors: M
authors: M
authors: Vandevenne
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology; D001667:Binding, Competitive; D008958:Models, Molecular; D009113:Muramidase / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; C421864:hen egg lysozyme; D009113:Muramidase; D001618:beta-Lactamases
keywords: antibody engineering; multifunctional enzyme; nanobody; protein chimera; β-lactamase