Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock.

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.