Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-04-18 DOI:10.1016/j.pep.2024.106485

Aliasghar Rahimian , Ali Nabati , Hooman Askari , Mohammad Saffarioun , Mahdi Aminian

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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 (K_D) 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.

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用简单的生物信息学方法设计和构建噬菌体显示的驼科纳米抗体库

背景合成噬菌体显示文库的合理设计需要确定最合适的位置，使用定义的氨基酸组进行随机化，以再现自然发生。本研究使用位置特异性评分矩阵（PSSM）来识别和随机化驼科纳米抗体（VHH）CDR3。方法基于 PSSM 分析，确定了 cAbBCII10 VHH 框架的 CDR3，并定义了用于替换 PSSM-CDR3 位置的一组氨基酸。利用 Rosetta 设计 SwiftLib 工具，将最终序列反转译为退化核苷酸序列。结果构建了一个具有 1 × 108 个变体的合成噬菌体显示 VHH 库。从该文库中分离出了三种与 rfVII 结合的 VHH，其解离常数（KD）分别为 1 × 10-8 M、5.8 × 10-8 M 和 2.6 × 10-7 M。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.