Cloning, baculoviral expression, detergent optimization and purification of human serotonin 5-HT 2A G-protein-coupled re
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ORIGINAL ARTICLE
Cloning, baculoviral expression, detergent optimization and purification of human serotonin 5‑HT2A G‑protein‑coupled receptor Gopa Mahesh1,2 Received: 4 March 2020 / Revised: 22 June 2020 / Accepted: 18 July 2020 © Springer Nature Singapore Pte Ltd. 2020
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. Keyword Human 5-HT2A · Construct preparation · Sf9 cells · Detergent screening · Dot blot · n-Dodecyl-β-dmaltopyranoside Abbreviations Bluo-gal Halogenated indolyl galactoside BRIL Cytochrome b562 RIL BSA Bovine serum albumin CD Circular dichroism CHS Cholesteryl hemisuccinate CNS Central nervous system CV Column volume Da Dalton DAPI 4,6-Diamidino-2-phenylindole Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42485-020-00043-w) contains supplementary material, which is available to authorized users. * Gopa Mahesh [email protected]; [email protected] 1
Structural Biology and Bio‑Informatics Division, CSIRIndian Institute of Chemical Biology, Kolkata 700032, India
Research and Development Department, Sudershan Biotech Pvt Ltd, Hyderabad 500074, India
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DDM n-Dodecyl-β-d-maltopyranoside E. coli Escherichia coli GFP Enhanced green fluorescent protein GPCR G protein-coupled receptor hu5-HT2A Human 5-Hydroxytryptamine 2A receptor HEPES 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid IPTG Isopropyl β-d-1-thiogalactopyranoside Kb Kilobase MOI Multiplicity of infection PBS Phosphate buffered saline PDB Protein data bank Pfu Plaque forming units Sf9 Spodoptera frugiperda SFM Serum-free medium mg Milligram µg Microgram µM Micro molar hr Hour rpm Revolutions per minute β2-AR Beta 2-adrener
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