Functional GPCR expression in eukaryotic LEXSY system

Aleksandra Luginina; Ivan Maslov; Polina Khorn; Oleksandr Volkov; Andrey Khnykin; Pavel K. Kuzmichev; Mikhail Shevtsov; Anatoliy Belousov; Ivan Kapranov; Dmitrii Dashevskii; Daniil A. Kornilov; Ekaterina Bestsennaia; Johan Hofkens; Jelle Hendrix; Thomas Gensch; Vadim Cherezov; Valentin Ivanovich; Alexey Mishin; Valentin Borshchevskiy

doi:10.1016/j.jmb.2023.168310

Functional GPCR expression in eukaryotic LEXSY system

Aleksandra Luginina, Ivan Maslov, Polina Khorn, Oleksandr Volkov, Andrey Khnykin, Pavel K. Kuzmichev, Mikhail Shevtsov, Anatoliy Belousov, Ivan Kapranov, Dmitrii Dashevskii, Daniil A. Kornilov, Ekaterina Bestsennaia, Johan Hofkens, Jelle Hendrix, Thomas Gensch, Vadim Cherezov, Valentin Ivanovich, Alexey Mishin, Valentin Borshchevskiy^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

6 Citations (Scopus)

Abstract

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.

Original language	English
Article number	168310
Number of pages	16
Journal	Journal of Molecular Biology
Volume	435
Issue number	23
DOIs	https://doi.org/10.1016/j.jmb.2023.168310
Publication status	Published - 1-Dec-2023
Externally published	Yes

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Luginina, A., Maslov, I., Khorn, P., Volkov, O., Khnykin, A., Kuzmichev, P. K., Shevtsov, M., Belousov, A., Kapranov, I., Dashevskii, D., Kornilov, D. A., Bestsennaia, E., Hofkens, J., Hendrix, J., Gensch, T., Cherezov, V., Ivanovich, V., Mishin, A., & Borshchevskiy, V. (2023). Functional GPCR expression in eukaryotic LEXSY system. Journal of Molecular Biology, 435(23), Article 168310. https://doi.org/10.1016/j.jmb.2023.168310

@article{abab91fad1294c0194e4f3c1569247c0,

title = "Functional GPCR expression in eukaryotic LEXSY system",

abstract = "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.",

author = "Aleksandra Luginina and Ivan Maslov and Polina Khorn and Oleksandr Volkov and Andrey Khnykin and Kuzmichev, {Pavel K.} and Mikhail Shevtsov and Anatoliy Belousov and Ivan Kapranov and Dmitrii Dashevskii and Kornilov, {Daniil A.} and Ekaterina Bestsennaia and Johan Hofkens and Jelle Hendrix and Thomas Gensch and Vadim Cherezov and Valentin Ivanovich and Alexey Mishin and Valentin Borshchevskiy",

year = "2023",

month = dec,

day = "1",

doi = "10.1016/j.jmb.2023.168310",

language = "English",

volume = "435",

journal = "Journal of Molecular Biology",

issn = "0022-2836",

publisher = "Academic Press",

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}

Luginina, A, Maslov, I, Khorn, P, Volkov, O, Khnykin, A, Kuzmichev, PK, Shevtsov, M, Belousov, A, Kapranov, I, Dashevskii, D, Kornilov, DA, Bestsennaia, E, Hofkens, J, Hendrix, J, Gensch, T, Cherezov, V, Ivanovich, V, Mishin, A & Borshchevskiy, V 2023, 'Functional GPCR expression in eukaryotic LEXSY system', Journal of Molecular Biology, vol. 435, no. 23, 168310. https://doi.org/10.1016/j.jmb.2023.168310

TY - JOUR

T1 - Functional GPCR expression in eukaryotic LEXSY system

AU - Luginina, Aleksandra

AU - Maslov, Ivan

AU - Khorn, Polina

AU - Volkov, Oleksandr

AU - Khnykin, Andrey

AU - Kuzmichev, Pavel K.

AU - Shevtsov, Mikhail

AU - Belousov, Anatoliy

AU - Kapranov, Ivan

AU - Dashevskii, Dmitrii

AU - Kornilov, Daniil A.

AU - Bestsennaia, Ekaterina

AU - Hofkens, Johan

AU - Hendrix, Jelle

AU - Gensch, Thomas

AU - Cherezov, Vadim

AU - Ivanovich, Valentin

AU - Mishin, Alexey

AU - Borshchevskiy, Valentin

PY - 2023/12/1

Y1 - 2023/12/1

N2 - 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.

AB - 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.

U2 - 10.1016/j.jmb.2023.168310

DO - 10.1016/j.jmb.2023.168310

M3 - Article

SN - 0022-2836

VL - 435

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 23

M1 - 168310

ER -

Functional GPCR expression in eukaryotic LEXSY system

Abstract

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