A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system

• Published in: Protein expression and purification Vol. 215; p. 106392
• Main Authors: Wang, Yao, Weng, Shaoting, Tang, Yajie, Lin, Sen, Liu, Xiayue, Zhang, Wenhui, Liu, Gang, Pandi, Boomi, Wu, Yinrong, Ma, Lei, Wang, Lin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2024
• Subjects: Antigens, CD20 - genetics; Antigens, CD20 - metabolism; Claudins - metabolism; Escherichia coli - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rituximab - genetics; Rituximab - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2023.106392

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. •A CFPS method for producing hard-to-express four-pass transmembrane proteins was described.•Swapping the easier-to-express CD20's TMD with another transmembrane protein claudin 18.2 enhances the expression.•CD20's TMD may act an universal scaffold for presenting other four-pass tranmembrane proteins epitopes.