Resumen de Construction and validation of a large naïve library of VHHs integrated in the chromosome of E. coli for selection of nanobodies using bacterial display
In this PhD thesis, a large naïve library of nanobodies (Nbs) containing over a billion VHH sequences has been constructed and integrated into the E. coli chromosome for Nb surface display. This library is the largest collection of genes integrated in the chromosome of a living cell. Specific Nbs against the Ebola virus surface glycoprotein (GP-EBOV) and the mouse Programmed Dead-Ligand 1 (PD-L1) antigens were selected from the integrated naïve library using cell sorting methods, demonstrating its potential universality. The singularity of this Nb collection integrated in single copy in the bacterial chromosome may facillitate the in vivo affinity maturation of naïve Nbs by coupling in vivo mutagenesis strategies for Nb diversification with selection of binders for a broad range of applications in the medical, diagnostic and enviromental fields.
