Construct design, isolation and purification of the monomeric form of human GPCR GPR17 for structural and functional studies

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Resumo

G protein-coupled receptors (GPCRs) are a family of heptahelical transmembrane proteins consisting of more than 800 representatives in the human genome that regulate most processes in the human body and are targets for up to a third of all modern drugs. Many GPCRs, despite their importance for pharmacology, are still considered orphan, i.e., their endogenous ligand is unknown. Orphan receptor GPR17, belonging to class A GPCR, is expressed mainly in the central nervous system, plays an important role in the formation of the myelin sheath of neurons and is a potential target for the development of new drugs against multiple sclerosis, Alzheimer's disease and ischemia. The aim of this work was to prepare GPR17 for structure-functional studies, starting with the heterologous expression and ending with obtaining a stable protein sample. Screening of various genetically engineered constructs was performed, a number of point mutations were analyzed, and a significant number of potential ligands of this receptor were tested. As a result of the work, the conditions for expression, isolation, and purification of GPR17 were optimized, which together made it possible to obtain a fairly stable and monomeric protein preparation suitable for further structural studies.

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Sobre autores

N. Safronova

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Autor responsável pela correspondência
Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

A. Luginina

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

A. Sadova

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

M. Shevtsov

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

O. Moiseeva

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

V. Borshchevskiy

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

A. Mishin

Research Сenter for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology (National Research University)

Email: mishinalexej@gmail.com
Rússia, Dolgoprudny

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2. Additional materials
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3. Fig. 1. Results of gel filtration for the R252-BRIL-V253 construct with various point mutations and their combinations. SE – surface expression.

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4. Fig. 2. Plot showing the thermal shift resulting from point mutations introduced into GPR17. Average values ​​obtained from three independent experiments are shown.

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5. Fig. 3. Schematic representation of the best design. The introduced point mutations are shown in red, the partner protein BRIL is shown in purple, the HA fragment is shown in pink, the FLAG sequence is shown in yellow, 9 histidine residues are shown in blue, the TEV protease site is shown in orange, and atavistic linkers are shown in gray.

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6. Fig. 4. Normalized melting curve (a) and analytical gel filtration HPLC results (b) for the best construct R252-BRIL-V253 with mutations A1313.24L + Y1483.41W + F1583.51Y + F2315.49L + I2345.52L + A3297.57D. Solid line – sample with concentration of 0.1 mg/ml, dotted line – 30 mg/ml.

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7. Fig. 5. Effect of buffer salt composition on GPR17 thermal stability. Melting curves for several receptor constructs are shown. For each construct, the experiment was carried out in three different buffers: blue lines correspond to the buffer with NaCl in the composition, green lines – with KCl, and orange lines – with MgCl2. The salt concentration in this experiment was 500 mM.

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