Screening and functional studies of Norrin-related nanobodies

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-04-09 DOI:10.1016/j.pep.2025.106717

Yiwen Xu , Yiang Wang , Zhongyun Lan , Yaxin Tuo , Siyu Zhou , Shuaiying Zhao , Yunfeng Liu , Yingying Kong , Huarui Qiao , Jianfeng Xu , Yuanyuan Dai , Yong Geng

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引用次数: 0

Abstract

Norrin is a crucial regulator of the Wnt/β-catenin signaling pathway, playing a key role in retinal vascular development, blood-brain barrier maintenance, and neuroprotection. In this study, a Norrin fusion protein (Norrin-Fc) was successfully expressed and purified using an insect cell expression system. Camels were immunized with purified Norrin-Fc to generate nanobodies, which consist solely of the heavy chain variable domains of heavy chain antibodies (VHH) with a molecular weight of approximately 15 kDa. Two nanobody strains, Nb 1C4 and Nb 2B10, were identified for their specific binding to Norrin-Fc, and their binding affinities were further characterized. Flow cytometry analysis confirmed that Nb 1C4 and Nb 2B10 specifically bound to the Norrin-FZD4 fusion protein. Luciferase reporter assay results demonstrated that both nanobodies effectively disrupted LGR4-induced Wnt/β-catenin signaling upon Norrin stimulation. This study represents the first successful development of nanobodies targeting Norrin, providing a strong foundation for the advancement of Norrin-related diagnostic tools and antibody therapeutics.

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Norrin是Wnt/β-catenin信号通路的重要调节因子，在视网膜血管发育、血脑屏障维护和神经保护中发挥着关键作用。本研究利用昆虫细胞表达系统成功表达并纯化了 Norrin 融合蛋白（Norrin-Fc）。用纯化的 Norrin-Fc 对骆驼进行免疫以产生纳米抗体，纳米抗体仅由分子量约为 15 kDa 的重链抗体（VHH）的重链可变结构域组成。Nb 1C4 和 Nb 2B10 这两种纳米抗体菌株与 Norrin-Fc 有特异性结合，它们的结合亲和力也得到了进一步鉴定。流式细胞仪分析证实，Nb 1C4 和 Nb 2B10 能与 Norrin-FZD4 融合蛋白特异性结合。荧光素酶报告实验结果表明，这两种纳米抗体都能在诺林刺激下有效地破坏 LGR4 诱导的 Wnt/β-catenin 信号传导。这项研究首次成功开发了以Norrin为靶标的纳米抗体，为Norrin相关诊断工具和抗体疗法的发展奠定了坚实的基础。

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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.