Cell-free receptor-based biosensors

The ability to express and purify modified recombinant signalling proteins such that they retain their biological function in a cell-free context, has provided a basis for production of molecular biosensors. Here we utilise G-protein Coupled Receptors (GPCRs) and their G-proteins to detect various binding partners in a cell-free environment. Molecular biology approaches were employed to express these proteins using baculovirus and bacteria, and to alter their characteristics to improve surface-attachment and fluorescent labelling capabilities. Ligand-mediated signalling of a GPCR could be measured (using [³⁵S]GTPγS-binding assays) in a reconstituted system with recombinant proteins either free in solution or attached to Ni ²⁺-coated beads. Affinity of histidine-tagged proteins for a Ni ²⁺-coated surface was significantly enhanced by addition of extra histidine residues to the tag, as determined by surface plasmon resonance. This was due to the longer tag occupying, on average, a greater number of available histidine-binding sites. Further, a novel homogeneous fluorescence resonance energy transfer (FRET)-based assay has been developed to detect rearrangements in the G-protein heterotrimer. Investigation of small peptides that can be fused to G-protein subunits, allowing for site-specific fluorescent labelling, was undertaken in order to improve the resolution of the "first generation" FRET assay. By utilizing this improved G-protein heterotrimer "molecular switch", we are developing a generic technology such that a range of GPCRs could be assayed for ligand-mediated activation while attached to surfaces (e.g. on beads or as microarrays) or in solution (e.g. multi-well plates), with increased throughput.