Functional GPCR Expression in Eukaryotic LEXSY System

• Published in: Journal of molecular biology 2023-12, Vol.435 (23), p.168310-168310, Article 168310
• Main Authors: Luginina, Aleksandra, Maslov, Ivan, Khorn, Polina, Volkov, Oleksandr, Khnykin, Andrey, Kuzmichev, Pavel, Shevtsov, Mikhail, Belousov, Anatoliy, Kapranov, Ivan, Dashevskii, Dmitrii, Kornilov, Daniil, Bestsennaia, Ekaterina, Hofkens, Johan, Hendrix, Jelle, Gensch, Thomas, Cherezov, Vadim, Ivanovich, Valentin, Mishin, Alexey, Borshchevskiy, Valentin
• Format: Article
• Language: English
• Subjects: drugs; G protein-coupled receptors; genome; humans; insects; Leishmania tarentolae; molecular biology; Protein biophysical characterization; purinergic receptors; Recombinant expression; Single-molecule Förster Resonance Energy Transfer; superfamily
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2023.168310

[Display omitted] •Recombinant production is one of the bottlenecks in GPCR structural and functional investigations.•The efficacy of the eukaryotic system LEXSY for GPCR production was demonstrated on the human A2A adenosine receptor, as a model protein.•Side by side expression, purification and functional characterization was performed for the LEXSY and baculovirus (Sf9) expression systems.•The A2AAR expressed in Sf9 and LEXSY showed similar monodispersity, stability in apo and ligand-bound states, agonist-induced conformational changes and structural dynamics with 3.6-fold higher yield in case of the LEXSY sample. G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.