Expression and Purification of Transmembrane Domain of Insulin Receptors for Structural-Dynamic NMR Studies

Members of the insulin receptor family (InsR, IGF1R, and IRR) play a key role in metabolic regulation, yet the structural mechanisms governing their activation remain incompletely understood. While extracellular and kinase domain structures are well characterized, the transmembrane domains (TMDs), responsible for transmitting conformational changes, lack high-resolution structural data, particularly in their physiologically relevant dimeric states. This limitation impedes the development of targeted therapies for diseases like diabetes and cancer, where aberrant receptor signaling is a key driver. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying membrane proteins in near-native environments, offering insights into their dynamics and interactions. However, production of isotopically labeled TMDs for NMR studies remains challenging due to low expression yields and solubility issues. Here, we present an optimized cell-free continuous-exchange expression system for high-yield production of InsR, IGF1R, and IRR TMDs, coupled with efficient purification proved by heteronuclear NMR spectroscopy in a membrane mimicking environment.