Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
AryoGen Pharmed biopharmaceutical research center, Karaj, Iran.
Protein Expr Purif. 2024 Jul;219:106485. doi: 10.1016/j.pep.2024.106485. Epub 2024 Apr 18.
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).
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.
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.
PSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.
理性设计合成噬菌体展示文库需要使用定义的氨基酸集来识别最适合随机化的位置，以重现自然发生的情况。本研究使用位置特异性评分矩阵（PSSM）来识别和随机化骆驼科纳米抗体（VHH）CDR3。通过这种方法设计的合成 VHH 文库的功能已针对发现重组凝血因子 VII（rfVII）的新 VHH 结合物进行了测试。
基于 PSSM 分析，确定了 cAbBCII10 VHH 框架的 CDR3，并为每个 PSSM-CD3 位置的替换定义了一组氨基酸。使用 Rosetta 设计 SwiftLib 工具，最终的文库被回译为简并核苷酸序列。基于该文库构建了一个合成的噬菌体展示文库，并筛选抗 rfVII 的结合物。
构建了一个具有 1×10 个变体的合成噬菌体展示 VHH 文库。从该文库中分离出三个与 rfVII 结合的 VHH 结合物，其解离常数（K）估计为 1×10 M、5.8×10 M 和 2.6×10 M。
PSSM 分析是设计合成噬菌体展示文库的一种简单有效的方法。
