Enzymatic functionalization of a nanobody using protein insertion technology

in Protein Engineering, Design & Selection (2015), 28(10), 451-460

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 be- come a ... [more ▼]

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 be- come 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 consti- tutes a suitable technology to functionalize nanobodies and allowsthe creation of versatile tools that can be used in innovative biotechnological assays. [less ▲]

Chitosan-based biomimetic scaffolds and methods for preparing the same

Patent (2011)

The invention concerns chitosan-based biomimetic scaffolds and methods for modulating their intrinsic properties such as rigidity, elasticity, resistance to mechanical stress, porosity, biodegradation and ... [more ▼]

The invention concerns chitosan-based biomimetic scaffolds and methods for modulating their intrinsic properties such as rigidity, elasticity, resistance to mechanical stress, porosity, biodegradation and absorbance of exudates. Therefore, the present invention relates to a layered chitosan-based scaffold wherein said layered scaffold comprises at least two fused layers, wherein at least one layer consists of a chitosan nanofiber scaffold membrane and at least one of the other layers of a porous chitosan scaffold support layer. Moreover, the present invention provides a layered chitosan-based scaffold characterized by (i) a good adhesion between the porous and nanofiber layers, (ii) a tuneable porosity of the nanofiber layer by tuning the distance between the nanofibers, (iii) a stable nanofibers and porous morphology even when immersed in water or other solvents and a process for the preparation of such layered chitosan-based scaffold.Finally, the present invention provides the use of the layered electrospun chitosan-based scaffold of the invention or the layered electrospun chitosan-based scaffold produced by the process of the invention as a wound dressing, in tissue engineering or for biomedical applications. [less ▲]

Comparative functional analysis of the human macrophage chitotriosidase

in Protein Science : A Publication of the Protein Society (2011)