G-protein coupled receptors are the largest class of membrane proteins that encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function and are popular drug targets with more that 60% of marketed drugs acting on these receptors. This study aims to investigate the biomolecular properties of GPCRs that influence and drive high-level expression and plasma membrane localization in Baker’s yeast, Saccharomyces cerevisiae, using the G-protein coupled receptor family as an experimental model system.
We chose the pYES2 yeast expression plasmid to introduce target genes into yeast, tagged with a yeast optimized gene for green fluorescent protein (ye-GFP). pYES2 is a high-copy number 2-μm episomal expression vector with a multiple cloning region that facilitates gene incorporation when the appropriate restriction sites are used. Additionally, it permits galactose-inducible expression of proteins under the direction of the GAL1 promotor which was originally derived from yeast and can be induced up to 1000-fold in the presence of galactose. GFP was introduced to the pYES2 system, subcloned into XL1-Blue E. coli cells and transformants were selected for resistance to carbenicillin. The gene sequence of GFP in pYES2 was verified and the plasmid was transformed in S. cerevisiae. pYES2-yeGFP transformants were subsequently selected from “synthetic dropout” agar plates (SC-URA−) and colonies were propagated in SC-URA− broth and tested for expression using immunoblotting techniques and fluorescence microscopy.
This study aims to test the expression and cell membrane localization of GPCR chimera (Prepro-D2(N-term)-A2A) c-terminally tagged with GFP gene at different post induction time period, lower temperature (16 ºC, 21 ºC, 30 ºC) and minimal media using widefield fluorescence microscopy.
Although, the optimal window of GFP gene expression and chimera was found to be 48 hours post-induction in a galactose media, no expression was observed at 72-hour post induction. Expression at lower temperature didn’t significantly affect the expression pattern. Chimera didn’t localize to the cell membrane when expressed in yeast. Addition of Tunicamycin showed more localization to ER.
The study conducted here will uncover new strategies toward optimizing S. cerevisiae for high-level protein expression of important molecular targets and using the well-characterized pYES2 system further allows us to modulate properties such as promoter strength to further tune expression.
|Commitee:||Liebl, Faith, Wei, Chin-Chuan, Dixon, Robert|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 81/7(E), Masters Abstracts International|
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