Construct Design, Isolation, and Purification of the Human GPR17 Receptor Monomeric Form for Structural and Functional Studies

Abstract

Objective: G protein-coupled receptors (GPCRs) are a family of seven transmembrane domain proteins with more than 800 members in the human genome. They play a key role in the regulation of most of the processes in the human body and are the targets of one third of all modern drugs. Despite their importance in pharmacology, many GPCRs remain orphan receptors, i.e., their endogenous ligands are unknown. An orphan receptor GPR17, a class A GPCR representative, is predominantly expressed in the central nervous system and plays an important role in regulating the formation of the myelin sheath of neurons. It is a potential target for the development of new drugs against various disorders, such as multiple sclerosis, Alzheimer’s disease, and ischemia. The aim of this work was to prepare GPR17 for structural and functional studies starting from protein modification and ending with the receptor production. Methods: A screening of different genetically engineered constructs was performed, a series of point mutations were analyzed, and a significant number of potential ligands for this receptor were tested. The constructs were expressed in the Sf9 insect cell line using the Bac-to-Bac approach. The membrane fraction was extracted, the protein was solubilized into DDM/CHS detergent micelles, followed by purification by a metal affinity chromatography. Functional analysis of the purified protein included analytical gel filtration, polyacrylamide gel electrophoresis, and thermal stability analysis. Results and Discussion: Stabilizing point mutations were identified and the optimal position of the partner protein was found. The conditions for the expression, isolation, and purification of GPR17 were optimized to produce a sufficiently stable and monomeric protein sample suitable for further structural and functional studies. Conclusions: GPR17 is an orphan GPCR and the actively studied pharmacological target. The structural and functional investigation of this protein is a relevant problem for modern science and biomedicine. As a result of an in-depth study of GPR17, several stabilizing receptor modifications were developed, and optimal conditions for recombinant protein production were found. Our results can be used for further structural and functional studies of GPR17 and can serve as an example of a strategy that can be applied to perform investigation of other GPCRs.