High-purity preparation and biophysical characterization of OX40-TC in lipid nanodiscs

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2025-03-04 DOI:10.1016/j.pep.2025.106697

Hao Wang , Xiaohui Zhang , Cunlong Yin , Lu Liu , Liang Chen , Hengfang Tang , Bo Wu , Junfeng Wang

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引用次数: 0

Abstract

Tumor necrosis factor receptor superfamily member 4 (OX40) is essential for the activation and maintenance of T cell immune function because of its recognition and regulation in signal transduction, therefore understanding the structure and dynamics of the membrane protein OX40 in a near-native membrane environment is critical for elucidating its functions. However, efforts to prepare OX40 in a stable, functional form and reconstitute it into a membrane-mimicking lipid system face persistent challenges, limiting progress in its structural and functional characterization. Here, we developed an efficient method for the expression in E. coli and purification of the N-terminal truncated transmembrane and cytoplasmic domains of OX40 (OX40-TC) using a TrpLE fusion system. High-purity OX40-TC was achieved and successfully reconstituted into membrane scaffold protein (MSP)-nanodiscs for the first time. Characterization through size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy confirmed their stability and homogeneity. This study provides a reliable platform for investigating the structure and function of OX40-TC, advancing our understanding of OX40-mediated signaling and its therapeutic potential.

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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.