Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Bioengineering, Dalian University of Technology, Dalian, Liaoning, China.
Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium.
Methods Mol Biol. 2022;2446:357-371. doi: 10.1007/978-1-0716-2075-5_18.
Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.
纳米抗体（Nbs）可以通过噬菌体、细菌、酵母或核糖体展示免疫、合成或原始文库成功回收。然而，在经过淘选后，需要筛选和评估多个单个 Nb 克隆的可溶性、抗原特异性、亲和力和潜在生物学功能。因此，非常需要有一种方便的表达策略来获得足够的蛋白质，以深入表征 Nbs。在这方面，带有纯化和检测标签以及可反应的化学基团以方便地生成 Nb 衍生物将有很大帮助。在这里，我们提供了一种用于高产细胞质表达和纯化 FGE-标记 Nbs 的通用方案。FGE 标签中的半胱氨酸很容易通过使含有 FGE 标签的 Nb 通过连续流生物催化系统转化为甲酰甘氨酸。Nbs 末端的 formylglycine 侧链中的醛基适合用于随后的生物正交反应，与荧光染料、生物素、聚乙二醇或色谱树脂结合。我们还包括生产高产重组 FGE 的方法，以及将其固定在 Sepharose 上以产生连续流生物催化系统的条件。
