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The vomeronasal system is one of several fine-tuned scent-detecting signaling systems in mammals. However, despite significant efforts, how these receptors detect scent remains an enigma. One reason is the lack of sufficient purified receptors to perform detailed biochemical, biophysical and structural analyses. Here we report the ability to express and purify milligrams of purified, functional human vomeronasal receptor hVN1R1. Circular dichroism showed that purified hVN1R1 had an alpha-helical structure, similar to that of other GPCRs. Microscale thermophoresis showed that hVN1R1 bound its known ligand myrtenal with an EC[subscript 50] ∼1 µM. This expression system can enable structural and functional analyses towards understanding how mammalian scent detection works., United States. Defense Advanced Research Projects Agency (HR0011-09-C-0012)

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins., United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012), Massachusetts Institute of Technology. Undergraduate Research Opportunities Program