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.