Purification of CD40 signaling complex for structural study

Abstract

CD40-CD40L is a pair of Tumor Necrosis Factor Receptor (TNFR) and Tumor Necrosis Factor (TNF) superfamily. This interaction is generated in extracellular domain of CD40 and critical to immune system. They play a role for T-cell-mediated B-cell activation in immune response. Also, CD40 is able to bind with adaptor proteins on intracellular domain of CD40. It induces diverse cell signaling related to cell proliferation, cell death, survival, and differentiation. In this thesis, the full-length CD40 receptor was expressed successfully, and formation of human full length CD40-CD40L-TRAF2-Fab (CP-870,893) was also successfully made. For cryo-EM structural analysis, hundreds kDa size proteins are needed at least. In addition, Fab binding complex forms distinguishable shape in Cryo-EM images. Therefore, this complex is suitable for cryo-EM and further work helps structural studies of CD40-mediated signaling pathway.