Design and Construction of a Phage-Displayed Camelid Nanobody Library Using a Simple Bioinformatics Method Publisher Pubmed



Rahimian A¹ ; Nabati A¹ ; Askari H¹ ; Saffarioun M² ; Aminian M¹ Show Affiliations
Source: Protein Expression and Purification Published:2024

Abstract

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. © 2024 Elsevier Inc.