RESUMO
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 × 108 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (KD) of 1 × 10-8 M, 5.8 × 10-8 M and 2.6 × 10-7 M. CONCLUSION: PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.
