Liaoning Key Laboratory of Molecular Recognition and imaging, School of Bioengineering, Dalian University of Technology, No.2 Linggong Road, Dalian 116023, China.
Biomolecules. 2020 Nov 27;10(12):1610. doi: 10.3390/biom10121610.
VHH-based immunosorbents are an emerging and promising tool for the removal of toxic substances from plasma. However, the small size of VHHs is a double-edged sword, bringing both benefits and drawbacks to the immunosorbent. The small size of the VHH allows a higher coupling density, while the closer distance to the resin might create steric hindrance for paratope access. The latter could be avoided by inserting a linker between the VHH and the gel attachment site. Here, we report an approach to improve the activity retention of the immobilized VHH by selecting suitable linkers between the VHH and the site-specific immobilization site on the resin. Seven peptide linkers differing in length and flexibility were fused to the VHH and contained the formylglycine generating enzyme (FGE) recognition sequence. These constructs were expressed in the cytoplasm of bacteria and purified, the VHH production yield and affinity for its cognate antigen was measured. Furthermore, the fGly conversion, the immobilization of the aldehyde-containing nanobodies, the immobilization on resin and the antigen binding activity of the VHH-based immunoadsorbents was monitored. The VHH with longer and rigid, proline-rich linkers exhibited good expression yield of approximately 160 mg/L of culture, a fGly conversion of up to 100%, and the highest activity retention rate of more than 68%. This study unveiled two suitable linkers for the preparation of VHH-based immunosorbents that will assist the development of their clinical application.
基于 VHH 的免疫吸附剂是一种新兴的、有前途的工具，可用于从血浆中去除有毒物质。然而，VHH 的小尺寸是一把双刃剑，为免疫吸附剂带来了好处和缺点。VHH 的小尺寸允许更高的偶联密度，而与树脂的更近距离可能会对变构部位的进入造成空间位阻。通过在 VHH 和凝胶附着位点之间插入接头，可以避免这种情况。在这里，我们报告了一种通过在 VHH 和树脂上的特异性固定化位点之间选择合适的接头来提高固定化 VHH 的活性保留的方法。将七个在长度和灵活性上不同的肽接头融合到 VHH 上，并包含甲酰甘氨酸生成酶 (FGE) 识别序列。这些构建体在细菌的细胞质中表达并进行纯化，测量 VHH 的产量和对其同源抗原的亲和力。此外，还监测了 fGly 转化、含醛纳米体的固定化、醛的固定化以及基于 VHH 的免疫吸附剂的抗原结合活性。具有更长和刚性、脯氨酸丰富接头的 VHH 表现出约 160mg/L 培养物的良好表达产量、高达 100%的 fGly 转化率以及超过 68%的最高活性保留率。这项研究揭示了两种适合制备基于 VHH 的免疫吸附剂的接头，这将有助于它们的临床应用开发。
