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 the authors 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]GTPgammaS-binding assays) in a reconstituted system with recombinant proteins either free in solution or attached to Ni2+-coated beads. Affinity of histidine-tagged proteins for a Ni2+-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.Richard V. Glatz, Wayne R. Leifert, Kelly Bailey, Tamara H. Cooper, Chris S. Barton, A. Scott Martin, Amanda Aloia, Olgatina Bucco, Lakshmi Waniganayake, Gang Wei, Burkhard Raguse, Lech Wieczorek, and Edward J. McMurchi