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Updated: Jul 19, 2026
Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries
Published on: January 17, 2015
Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method
Aliasghar Rahimian1, Ali Nabati1, Hooman Askari1
1Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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.
