Screening and functional characterization of nanobodies targeting the transferrin receptor

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2025-03-13 DOI:10.1016/j.pep.2025.106702

Yuting Ding , Li Jia , Qifubo Geng , Yan Liu , Shaojue Guo , Shuaiying Zhao , Yingying Kong , Quanfang Jin , Guangxu Xu , Jianfeng Xu

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Abstract

The transferrin receptor (TfR1) mediates the cellular uptake of iron and other molecules, playing a vital role in hematology and tumor growth. Nanobodies (NBs) targeting TfR1 offer promising therapeutic potential due to their small size, high specificity and stability. However, rapid identification of effective nanobodies remains challenging.In this study, the truncated extracellular fragment of human TfR1 was expressed in a prokaryotic system and purified. Immunized camelids provided a source for nanobody libraries, which were screened using phage display and high-throughput strategies to identify candidates with specific TfR1 binding.NB 2D7 with nanomolar-level dissociation constants (KD) were successfully identified.The analysis of Cell Counting Kit-8(CCK8) experiments indicates that the combined treatment of NB2D7 with FeCl₃ can reduce the survival rate of LoVo cells.This research establishes an efficient platform for anti-TfR1 nanobody screening and highlights the therapeutic potential of these nanobodies in cancer treatment and iron metabolism disorders.

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转铁蛋白受体靶向纳米体的筛选与功能表征。

转铁蛋白受体（TfR1）介导铁和其他分子的细胞摄取，在血液学和肿瘤生长中起着至关重要的作用。靶向TfR1的纳米体（NBs）由于其体积小、特异性高和稳定性好，具有良好的治疗潜力。然而，快速鉴定有效的纳米体仍然具有挑战性。在本研究中，截断的人TfR1细胞外片段在原核系统中表达并纯化。免疫后的骆驼提供了纳米体文库的来源，利用噬菌体展示和高通量策略筛选具有特异性TfR1结合的候选物。成功地鉴定了具有纳米级解离常数（KD）的NB 2D7。细胞计数试剂盒-8（CCK8）实验分析表明，NB2D7与FeCl3联合处理可降低LoVo细胞的存活率。本研究建立了抗tfr1纳米体筛选的有效平台，突出了这些纳米体在癌症治疗和铁代谢紊乱中的治疗潜力。

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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.