Molecular engineering of G protein-coupled receptors and G proteins for cell-free biosensing

The ability to express and purify modified recombinant proteins, so they retain their biological function in a cell-free format, has provided a basis for development of molecular biosensors. Here we utilize recombinant G protein-coupled receptors (GPCRs) and their G proteins for cell-free detection of various binding partners. Fusion peptides were used to improve surface-attachment and fluorescent-labelling capabilities. A novel homogeneous fluorescence resonance energy transfer (FRET)-based assay was developed to detect rearrangements in the G protein heterotrimer. By using this heterotrimeric 'molecular switch', we are developing a generic technology such that multiple GPCRs could be assayed for ligand-mediated activation while tethered to surfaces or in solution, with increased throughput compared to current assay platforms.