Structure-based design and construction of a synthetic phage display nanobody library

Objective: To design and construct a new synthetic nanobody library using a structure-based approach that seeks to maintain high protein stability and increase the number of functional variants within the combinatorial space of mutations. Results: Synthetic nanobody (Nb) libraries are emerging as an attractive alternative to animal immunization for the selection of stable, high affinity Nbs. Two key features define a synthetic Nb library: framework selection and CDR design. We selected the universal VHH framework from the cAbBCII10 Nb. CDR1 and CDR2 were designed with the same fixed length as in cAbBCII10, while for CDR3 we chose a 14-long loop, which creates a convex binding site topology. Based on the analysis of the cAbBCII10 crystal structure, we carefully selected the positions to be randomized and tailored the codon usage at each position, keeping at particular places amino acids that guarantee stability, favoring properties like polarity at solvent-exposed positions and avoiding destabilizing amino acids. Gene synthesis and library construction were carried out by GenScript, using our own phagemid vector. The constructed library has an estimated size of 1.75 × 10⁸. NGS showed that the amino acid diversity and frequency at each randomized position are the expected from the codon usage.