Cloning, baculoviral expression, detergent optimization and purification of human serotonin 5-HT2A G-protein-coupled receptor

Abstract

The serotonin 5-HT2A receptor is a member of G-protein-coupled receptor (GPCR) superfamily. Human 5-HT2A (hu5-HT2A) receptor is broadly distributed in the central nervous system (CNS) and is responsible for many functions including learning and memory. Hence, many psychiatric disorders are linked with altered 5-HT2A receptor activities, and it remains one of the encouraging drug targets for several mental disorders. In addition, antagonists of this receptor have antipsychotic and antidepressant properties, while agonists maintain cognition-build up and hallucinogenic properties. GPCRs are membrane proteins, associating strongly with membrane lipid bilayers by hydrophobic interactions. Thus, their purification is technically challenging. By their hydrophobic nature, GPCRs are especially inclined to form insoluble aggregates some time after their separation from the cell membrane. To prevent nonspecific aggregation, identification of a suitable detergent is important for use in the purification process and to help protein to remain in soluble and monodispersed condition. This study involved preparation of gene constructs of native hu5-HT2A GPCR for over-expression in Sf9 cells, followed by solubilization of Sf9 cells using an array of detergents, and finally native receptor was partially purified using optimal detergent n-dodecyl-β-d-maltopyranoside.