分化簇 20 (CD20) 是一种非糖基化、多跨跨膜蛋白，由 B 淋巴细胞特异性整合。与 CD20 类似，另一种四次跨膜蛋白 Claudin 18.2 作为新兴的癌症治疗靶点引起了人们的关注。然而，它们的溶解度差和毒性往往阻碍下游应用，例如抗体药物的开发。因此，开发一种经济有效的方法来生产具有多个跨膜域的药物靶点至关重要。在这项研究中，通过基于大肠杆菌的体外转录-翻译偶联系统实现了高产率的重组CD20。表面等离子共振结果表明，利妥昔单抗（一种抗白血病药物）与 CD20 蛋白具有纳摩尔级亲和力，这与已发表的结果一致。值得注意的是，通过用 CD20 的跨膜结构域替换其跨膜结构域，以前难以表达的紧密蛋白 18.2 重组蛋白在同一反应系统中成功表达。使用商业抗密蛋白 18 抗体验证嵌合蛋白胞外结构域的折叠。该研究为促进四次跨膜蛋白的表达提供了新的概念，为膜相关药物靶点的大规模工业化生产奠定了基础，类似于claudin 18.2。版权所有©2023。由Elsevier Inc.出版。

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli-based in vitro coupled transcription-translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.Copyright © 2023. Published by Elsevier Inc.