Cloning and Expression of Epithelial Sodium Channels in Saccharomyces Cerevisiae
Date
2007-12
Authors
Boswell, Mikki
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Abstract
Epithelial Sodium Channels (ENaC) are integral membrane proteins which regulate sodium re-absorption within cells. Current knowledge about these channels is limited and includes physiological functional studies involving the investigation of cellular responses upon activation and inhibition of these channels. The subunit composition of this particular channel is unknown, and researchers currently debate on whether functional ENaC is a tetramer, with 2-a, 1-~, and 1-y ENaC, or larger multimeric complex composed of equal numbers of each subunit. Knowledge about the number and association of subunits as well as a crystal structure would give researchers the ability to study interactions between the protein's subunits and make predictions in how alterations in structure might affect the protein's function. My research will aid in determining ENaC's structure by increasing the amount of expressed protein available for future studies. By cloning ENaC into Saccharomyces cerevisiae, we will be able to overcome the limitations of low protein expression seen with traditional mammalian expression systems currently used. Thus, researchers will have the necessary information to carry out studies identifying structural features and further explaining the physiological role in animals.
For this study, mammalian expression vectors containing each ENaC gene were used as template in PCR to amplify each of the ENaC genes. The three ENaC genes were cloned into separate yeast vectors and transformed into competent S. cerevisiae cells for expression. The ENaC subunits were expressed individually and in combination in yeast cells as seen by western blot analysis. Expression conditions were optimized using time, sugar source, and various yeast strains. In all strains induction times of ~4 hours result in maximum expression when raffinose was used to replace glucose, expression of alpha ENaC in the yeast strain SllnsE4A S. cerevisiae resulted in a significant increase in protein yield. In this study, we have demonstrated that ENaC expression was achieved at the microgram level, which will provide adequate quantities that can ultimately be used to achieve mass spectroscopy analysis and gain adequate knowledge into the stoichiometry of the ENaC subunits.
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Keywords
membrane proteins, molecular cloning, saccharomyces cerevisiae, sodium channels, ion channels
Citation
Boswell, M. (2007). Cloning and expression of epithelial sodium channels in Saccharomyces cerevisiae (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.