An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions

化学 洗脱 色谱法 亲和层析 配体(生物化学) 离解常数 吸附 选择性 重组DNA 生物化学 有机化学 催化作用 受体 基因
作者
Jun Ren,Hao Xiong,Chundong Huang,Fangling Ji,Lingyun Jia
出处
期刊:Journal of Chromatography A [Elsevier BV]
卷期号:1676: 463274-463274 被引量:4
标识
DOI:10.1016/j.chroma.2022.463274
摘要

Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application. Aiming to improve elution efficiency, structure-guided mutations of BC2-Nb were conducted to adjust the structural flexibility of its antigen-binding site. Six ligand variants were obtained with their binding affinity decreasing by about 100-fold. Among them, one mutated BC2-Nb named 44D was chosen to prepare immunoaffinity resin, and its adsorption and elution performance were well characterized. The site-directed mutation led to the equilibrium dissociation constant (KD) of BC2-Nb changing from 1.4 × 10-9 M to 1.4 × 10-7 M (44D). The resin using 44D as ligand retained a static binding capacity of 19.14 mg/mL toward BC2T-fused enhanced green fluorescent protein (eGFP-BC2T). Significantly improved elution efficiency was obtained with the mutated ligand. Protein recovery reached 94% at pH 3.5 for 44D-based resin, while the resin based on original BC2-Nb could only achieve its highest recovery of 84% at pH 2. In addition, a neutral elution condition (1 M arginine containing 50% propylene glycol, pH 7.4) was also found effective, which allowed a product recovery of 95%. The resin enabled direct capturing of eGFP-BC2T from bacterial lysates, and the one-step purification with the both elution conditions could achieve a product purity of more than 90%. This study provided a promising affinity ligand, and also proved the feasibility of controlling the elution process of nanobody-based affinity resin through the strategy of binding sites modification.
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