Event
ChBE Seminar Series: Anne Skaja Robinson
Tuesday, February 26, 2013
11:00 a.m.
Room 2108 Chemical and Nuclear Engineering Bldg.
Professor Ganesh Sriram
gsriram@umd.edu
Characterizing and re-engineering yeast expression of G-protein coupled receptors: Lighting the best path for stability and biophysical characterization
Anne Skaja Robinson
Professor and Chair
Department of Chemical and Biomolecular Engineering
Tulane University
Proteins that reside in the cell membrane represent the most difficult challenges for expression and isolation, because they are partially hydrophobic, flexible, and unstable in isolation. However, they are among the most important of all proteins, as they play key roles in almost every cellular process, and represent over a third of all proteins. Here, I will highlight our studies of expression G-protein coupled receptors (GPCRs), in which we try to understand how the membrane proteins form and insert into the membrane of our heterologous host, S. cerevisiae, as well as characterize their properties once they are isolated in membrane-mimetic environments.
Over the last eight years, we have had great success expressing the human adenosine A2a (A2aR) in yeast. Here, typical yields were ~ 7 mg active A2aR per liter of culture; activity was verified by ligand-binding assays. Because of the ability to purify mg quantities of active protein, we have been able to carry out biophysical studies to understand the role of detergent and the disulfide bond network on the protein stability.
In addition, we have been examining the limitations to high-level production of other GPCRs. Our recent studies have focused on identifying the cellular localization of the retained protein, and by using chimeric GPCRs to test the role of potential trafficking motifs on protein retention, trafficking, and activity. Through this combination of cellular and in vitro approaches, we can better understand the limitations to large-scale production and biophysical studies of the G-protein-coupled receptors the largest known protein superfamily, and the targets of 30-50% of drug discovery efforts.
