Functional GPCR Expression in Eukaryotic LEXSY System

• DOI: 10.1016/j.jmb.2023.168310
• Publication Date: 2023-10-07T00:25:23Z
• Authors: Aleksandra Luginina; Ivan Maslov; Polina Khorn; Oleksandr Volkov; Andrey Khnykin; Pavel Kuzmichev; Mikhail Shevtsov; Anatoliy Belousov; Ivan Kapranov; Dmitrii Dashevskii; Daniil Kornilov; Ekaterina Bestsen...; Johan Hofkens; Jelle Hendrix; Thomas Gensch; Vadim Cherezov; Valentin Ivanovich; Alexey Mishin; Valentin Borshche...
• Keywords: STRUCTURAL BASIS; LIGAND RECOGNITION; Biochemistry & Molecular Biology; Protein biophysical characterization; Receptor, Adenosine A2A; Protein Conformation; Ligands; Receptors, G-Protein-Coupled; Leishmania tarentolae; Recombinant expression; PROTEIN-COUPLED RECEPTORS; G protein-coupled receptors; ADENOSINE A(2A) RECEPTOR; BINDING; Drug Discovery; Humans; Leishmania; Science & Technology; COMPLEX; MEMBRANE-PROTEINS; Protein Stability; SINGLE-MOLECULE FRET; Recombinant Proteins; ALLOSTERIC REGULATION; Single-molecule Forster Resonance Energy Transfer; Life Sciences & Biomedicine

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.