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Cloning and Expression of Epithelial Sodium Channels (ENaC) in Saccharomyces cerevisiae
Author(s) -
Boswell Mikki Gage,
Booth Rachell E
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.813.11
Subject(s) - epithelial sodium channel , saccharomyces cerevisiae , cloning (programming) , yeast , chemistry , protein subunit , integral membrane protein , biochemistry , sodium , membrane protein , gene , membrane , organic chemistry , computer science , programming language
Epithelial Sodium Channels (ENaC) are integral membrane proteins which regulate sodium re‐absorption within cells. Current knowledge about the shoichiometry of these channels is limited; although a trimeric structure has recently been accepted. 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 which occur in traditional mammalian systems. For this study, the three ENaC genes were cloned into yeast vectors and transformed into S. cerevisiae cells. Expression conditions were optimized using time, sugar source, and various yeast strains. In all strains, induction times of approximately 4 hours resulted in maximum expression when raffinose was used as the sugar source, and expression of alpha ENaC in the yeast strain S1InsE4A 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 for mass spectroscopy analysis of ENaC's post‐translational modifications and subunit associations. Funding for this project has been provided by the Department of Chemistry and Biochemistry Texas State University‐San Marcos, Welch Foundation, and Research Corporation.