Expression and purification of an activated orexin receptor 1- G-protein complex

Orexin receptors constitute a family of class A G-protein coupled receptors. There are two subtypes of orexin receptors, namely OX1R and OX2R. OX1R and OX2R are widely distributed in the central nervous system and are the targets for the peptide neurotransmitters orexin-A and orexin-B. Orexins are involved in a plethora of key physiological functions such as regulation of the sleep/wake cycle, feeding behavior, energy homeostasis, and cognition. Dysfunction of the orexin system has been linked to various pathological conditions, such as narcolepsy, insomnia, obesity, addiction, cognitive impairment, and depression. The active state structure of OX2R has been elucidated, while the active state structure of OX1R remains unresolved. Here, we describe a method for the expression and purification of an activated OX1R bound to its native peptide ligand, orexin-A, in complex with a Dominant Negative Gsq protein and Nb35. The proteins were expressed in Hi5 insect cells and subsequently purified via two consecutive affinity chromatography steps, followed by a final polishing Size Exclusion Chromatography step. This study could stimulate further research into the activation mechanisms of OX1R and the structural determination of its active state structure.