ThermoBRET: a ligand-engagement nanoscale thermostability assay applied to GPCRs

Abstract

Sensitive assays to measure the thermostability of membrane proteins are important tools in protein purification optimisation and drug discovery. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptor 2 (CB2) as an example. This method applies the principles of Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that covalently binds cysteines in the GPCR transmembrane domain, exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations, revealing differences in thermostability for different detergent solubilising conditions and in the presence of stabilising ligands. In addition, we extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 °C vs 59 °C). ThermoBRET allows the high-throughput determination of GPCR thermostability which will be useful for protein purification optimisation strategies and as part of a drug discovery screening platform.

Sunlight‐Controllable Biopharmaceutical Production for Remote Emergency Supply of Directly Injectable Therapeutic Proteins

“…After transfection of the GLP‐1 encoding constructs in our 1.1E7 TRPV1 line, we first verified the correct function of the cells by measuring the concentration of the NLuc reporter before and after stimulation (Figure 3B). The special thermostable nanoluciferase (thermoLuc) reporter gene [ 28 ] encoded by the pBS1081 construct was confirmed to be present only in the transfected cells and was inducibly secreted upon activation of the cells (Figure 3C). Additionally, we also verified that the GLP‐1 peptide is expressed and that GLP‐1 secretion is induced by depolarization of the cells (Figure 3D).…”

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2

“…CXCR1 (GenBank: NM_000634.3) and CXCR2 (GenBank NM_001557.3) receptor cDNA sequences were amplified via a polymerase chain reaction and cloned downstream of a SNAP-tag (New England Biolabs, Hitchin, UK) cDNA sequence, between EcoRI and XhoI sites, in the previously generated pcDNA3.1neo(+)-SNAP mammalian expression vector (Invitrogen, Paisley, UK) 72 containing a Kozak sequence (GCCACC) and the 5-HT3 receptor signal sequence (amino acids MRLCIPQVLLALFLSMLTGPGEGSRK) upstream to facilitate membrane integration. Addition of either a LgBiT fragment 62 or thermostable Nanoluciferase (tsNanoLuc) 73 at the C terminus was achieved through cloning of corresponding sequences, in frame with receptor C termini, between XhoI/XbarI restriction sites, generating p3.1neo-SNAP-CXCR1/2-LgBiT and p3.1neo-SNAP-CXCR1/2-tsNanoLuc constructs. Constructs were used to generate HEK 293 stable cell lines through transfection using Lipofectamine 3000 (Invitrogen, US) in Opti-MEM media (Sigma Aldrich) with pcDNA3.1 SNAP-CXCR2-LgBit, and pcDNA3.1zeo-β-arrestin2-SmBit (GenBank NC_000017.1) 74 for NanoBiT complementation or pcDNA3.1neo-SNAP-CXCR1/CXCR2-tsNanoLuc (HEK 293 SNAP-CXCR2-NanoLuc or SNAP-CXCR1-NanoLuc) for NanoBRET binding and imaging assays.…”

Development of fluorescent peptide G Protein Coupled Receptor activation biosensors for NanoBRET characterisation of intracellular allosteric modulators

“…Original SNAP tag and Nanoluciferase sequences were from NEB (Hitchen UK) and Promega (Southampton UK) respectively. The tsNluc contains structural stabilising Nluc substitutions as described in Hoare et al 28,29 . Stable mixed population cell lines were established through G418 resistance (encoded by the plasmid vector (pcDNA3.1 neo + , Invitrogen, Paisley UK).…”