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

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.