Molecular Engineering of G Protein-Coupled Receptors and G Proteins for Cell-Free Biosensing

Richard V. Glatz; Wayne R. Leifert; Tamara H. Cooper; Kelly Bailey; Chris S. Barton; A. Scott Martin; Amanda L. Aloia; Olgatina Bucco; Lakshmi Waniganayake; Gang Wei; Burkhard Raguse; Lech Wieczorek; Edward J. McMurchie

doi:10.1071/CH06435

RESEARCH FRONT

Molecular Engineering of G Protein-Coupled Receptors and G Proteins for Cell-Free Biosensing

Richard V. Glatz ^A ^C , Wayne R. Leifert ^A , Tamara H. Cooper ^A , Kelly Bailey ^A , Chris S. Barton ^B , A. Scott Martin ^B , Amanda L. Aloia ^A , Olgatina Bucco ^A , Lakshmi Waniganayake ^B , Gang Wei ^B , Burkhard Raguse ^B , Lech Wieczorek ^B and Edward J. McMurchie ^A

+ Author Affiliations

- Author Affiliations

^A CSIRO Molecular and Health Technologies, Adelaide, SA 5000, Australia.

^B CSIRO Industrial Physics, Lindfield, NSW 2070, Australia.

^C Corresponding author. Email: glatz.richard@saugov.sa.gov.au

Australian Journal of Chemistry 60(5) 309-313 https://doi.org/10.1071/CH06435
Submitted: 16 November 2006 Accepted: 26 March 2007 Published: 28 May 2007

Abstract

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.

Acknowledgments

Baculovirus stocks (hexahistidine-tagged Gα_i1 and Gγ₂, and non-tagged α_2A adrenergic receptor) were obtained from Professor Richard Neubig, University of Michigan, USA. Gβ₄ baculovirus stock was obtained from Professor James Garrison, University of Virginia, USA. Complementary DNA for the chimeric Gα_16/s subunit was kindly supplied by Professor Yung-Hou Wong of the Hong Kong University of Science and Technology. FlAsH was synthesized and supplied by the Molecular Engineering group from CSIRO Molecular and Health Technologies, Clayton, Australia. This research was supported by the CSIRO’s Emerging Science Area for Nanotechnology funding scheme, as well as OzNano2Life (CG060027), which is a project proudly supported by the International Science Linkages program established under the Australian Government's innovation statement ‘Backing Australia's Ability.’

References

Molecular Engineering of G Protein-Coupled Receptors and G Proteins for Cell-Free Biosensing

Abstract

Acknowledgments

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