The serotonin 2A receptor (5-HT 2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT 2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT 2A R has marked distinction with ligand-bound conformations of 5-HT 2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT 2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier. K E Y W O R D S 5-HT 2A GPCR, baculoviral expression system, homology modeling, molecular dynamics simulation, protein over-expression and purification

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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.

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