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Review
. 2020 Mar:167:105524.
doi: 10.1016/j.pep.2019.105524. Epub 2019 Oct 31.

Expression and purification of recombinant G protein-coupled receptors: A review

Daniel N Wiseman  1 , Abigail Otchere  2 , Jaimin H Patel  3 , Romez Uddin  4 , Naomi L Pollock  5 , Sarah J Routledge  6 , Alice J Rothnie  7 , Cathy Slack  8 , David R Poyner  9 , Roslyn M Bill  10 , Alan D Goddard  11
Affiliations
Review

Expression and purification of recombinant G protein-coupled receptors: A review

Daniel N Wiseman et al. Protein Expr Purif. 2020 Mar.

Abstract

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

Keywords: Expression; GPCR; Purification; Review; SMALP.

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Figures

Fig. 1
Fig. 1
Ligand induced activation of a G protein-coupled receptor (GPCR). GPCRs (blue) are transmembrane receptors which activate intracellular signalling pathways, through coupling to G proteins. These heterotrimeric proteins consist of three subunits denoted α, β and γ, and are classically activated by a ligand induced conformational change in the GPCR [2]. This movement is proposed to involve a rotational transmembrane helix reorientation, exposing an intracellular binding cleft [3]. GDP is exchanged for GTP on Gα, while the βγ complex splits away and is able to signal independently of the Gα subunit. Humans encode 18, 5 and 12 different α, β and γ subunits, respectively. These combine into a variety of stimulatory (Gs) or inhibitory (Gi/q) effects on pathways including those dependent on adenylyl cyclase and phospholipase C. Created with Biorender.com.
Fig. 2
Fig. 2
The Drosophila GAL4-UAS system for targeted gene expression. The GAL4-UAS system can be used for targeted expression of GPCRs within the Drosophila photoreceptor cells (PRCs). To obtain flies expressing a gene of interest in a tissue specific pattern, two Drosophila strains are mated together in the parental cross. The driver strain expresses the yeast GAL4 protein from a tissue-specific enhancer/promoter. The UAS strain contains the gene of interest cloned downstream of the GAL4 Upstream Activating Sequence (UAS). The resulting F1 offspring will express GAL4 protein in a tissue-restricted pattern which will bind to the UAS sequences upstream of the gene of interest to drive its expression in those specific cells. By using a driver strain that expresses GAL4 specifically within the fly eye, heterologous GPCR expression can be restricted to the PRCs.
Fig. 3
Fig. 3
Solubilised GPCR in a detergent micelle compared to a SMALP. These diagrams show the interactions of the phospholipid bilayer (grey) or detergent monomers (red) with a GPCR (blue). Importantly, the difference between a detergent micelle and SMALP is shown, with regards to the retention of the GPCR's annular lipids when surrounded by the SMA co-polymer (purple). Created with Biorender.com.
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