Membrane proteins are an elusive class of biomolecules encompassing transporters, receptors, and enzymes. They are central to proper cell function and are also popular drug targets because they control processes like signaling, which can affect the onset and progression of diseases. Their structural characterization is critical to rational drug design and discovery, but is limited by difficulties in producing high-quality material in sufficient quantities. We aim to investigate the biomolecular properties of membrane proteins that influence and drive high-level expression and cell membrane targeting in Baker’s yeast, Saccharomyces cerevisiae, using the G-protein coupled receptor family as an experimental model system. Using the pYES2 episomal expression plasmid as a vehicle to introduce target genes into yeast, we have developed a system to monitor membrane protein expression by incorporating a green fluorescent protein (ye-GFP) tag at the C-terminus of our target. GFP was introduced to the pYES2 system, subcloned into XL1-Blue E. coli cells, transformants were selected based on their antibiotic resistance and the gene sequence of GFP in pYES2 was verified. We grew our yeast transformants in rich media containing galactose and expression of GFP was verified using a combinatorial approach involving western blot-based techniques and fluorescence microscopy. This analysis will facilitate new strategies toward optimizing S. cerevisiae for high-level protein expression of important molecular targets. It further allows us to use the pYES2 system as a scaffold to modulate properties such as promoter strength while developing new molecular cloning tools.

Developing molecular tools for membrane protein expression in Saccharomyces cerevisiae

2019 Spring National Meeting

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