Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method.

Background: Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII).

Methods: Based on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders.

Results: A synthetic phage-displayed VHH library with 1 × 10 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (K) of 1 × 10 M, 5.8 × 10 M and 2.6 × 10 M.

Conclusion: PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.