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Design of a phage-displayed Camelidae nanobody library using a simple bioinformatics method: Application of binders in immunoaffinity purification of recombinant coagulation factor VII
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  • Aliasghar Rahimian,
  • Ali Nabati,
  • Hooman Askari,
  • Mohammad Saffarioun,
  • Mahdi Aminian
Aliasghar Rahimian
Tehran University of Medical Sciences School of Medicine
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Ali Nabati
Tehran University of Medical Sciences School of Medicine
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Hooman Askari
Tehran University of Medical Sciences School of Medicine
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Mohammad Saffarioun
AryoGen Biopharma Co
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Mahdi Aminian
Tehran University of Medical Sciences School of Medicine

Corresponding Author:[email protected]

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Abstract

Background: Rational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using the best-fitting amino acid set to recapitulate the natural occurrence. To this end, the present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDRs. The functionality of a synthetic VHH repertoire designed by this method was tested against recombinant coagulation factor VII (rfVII). This study investigates the applicability of anti-rfVII VHH binders isolated from a PSSM-based library as immunoaffinity purification ligands. 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-CDR position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. This library was screened against recombinant coagulation factor VII, and the isolated VHH binders being coupled with Sepharose CL-4B beads were tested for rfVII purification. Results: A synthetic phage-displayed VHH library with 108 variants was constructed. Three VHH binders were isolated from this library with affinity constants (KAff)) of 108, 1.7x107, and 3.8x106. We could develop an immunoaffinity chromatography resin from one of the isolated binders. This resin showed acceptable specificity and stability with a capacity of 2.3 mg rfVII per ml of the resin. Conclusion: PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries. VHH binders discovered through this method can be effectively exploited in developing immunoaffinity chromatography resins.