Purification of the alpha subunit of the epithelial sodium channel (αENaC) for Surface Plasmon Resonance (SPR) studies
Date
2016-12
Authors
Berman, Chance N.
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Abstract
The epithelial sodium channel (ENaC) is responsible for sodium reabsorption in the distal convoluted tubules of the nephron in the kidneys. ENaC is a transmembrane protein with N and C-termini located in the cytosol and a larger extracellular loop containing a wrist, palm, beta-ball, thumb, knuckle and finger. Due to ENaCs hydrophilic and hydrophobic nature, isolation and purification while still being functional is not well understood. A 6x histidine tag was engineered into the extracellular loop of αENaC,
referred to hereon as αHis2-αENaC, and then was subcloned in the yeast expression vector pYES2/NTA. pYES2/NTA/αHis2-αENaC was transformed into S1, MATα ura3-52
leu2-3,112 trp1-289 his7-2 ade5-1 lys2::InsE-4A, and BY4742, MATα his3∆1 leu2∆ lys2∆ ura3∆ ygr204::kanMX4, yeast strains. Serial dilution assays, time-course expression trials, immobilized metal affinity chromatography (IMAC) and western blot analysis were employed to purify and verify expression of αHis2 in each strain of Saccharomyces cerevisae. ENaC peptides of each subunit were previously engineered and subcloned in the bacterial expression vector pGEX-4T-2. Immobilized substrate affinity chromatography (ISAC) was employed to purify peptides. Surface Plasmon Resonance (SPR) was conducted between ENaC peptides and whole subunit αHis2-αENaC. We report that the interactions of the palm and thumb region of βENaC, independently, between the whole αENaC subunit gave kinetic constants in the non-covalent range, potentially being an inter-subunit binding domain. Additional studies would use longer peptides, thought to be portions/ whole binding domains, to further elucidate inter- subunit binding regions.
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Keywords
Epithelial sodium channel, Surface Plasmon Resonance
Citation
Berman, C. N. (2016). <i>Purification of the alpha subunit of the epithelial sodium channel (αENaC) for Surface Plasmon Resonance (SPR) studies</i> (Unpublished thesis). Texas State University, San Marcos, Texas.