Expression, purification and characterization of SORCS2 intracellular domain for structural studies

Neurotrophin signaling pathways are one of the major cascades in neuronal development and involved in many key processes including proliferation, differentiation, apoptosis, synaptic plasticity, axonal growth. In addition to the main classes of neurotrophin receptors, Trk and P75NTR, there are many auxiliary proteins, which can also bind neurotrophins and regulate the signaling pathways. The versatility of interactions between them could explain multiple and completely opposite biological outcomes such as cell survival or apoptosis. Membrane protein SorCS2, a vacuolar protein sorting 10 protein-domain receptor, interacts with P75NTR and controls the activity of Trk receptors. The abnormal functioning of SorCS2 is associated with neurodegenerative diseases, such as Alzheimer's and Huntington's disease. But the mechanism of SorCS2 activation and basis of the interaction with P75NTR has remained elusive. Herein, we describe two efficient approaches for the intracellular domain of the SorCS2 production employing bacterial and cell-free expression systems, as well as purification and refolding protocols. Finally, we characterized the purified protein by DLS and NMR and demonstrated that the protein sample is suitable for structural studies.