title: Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.
abstract: The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αβγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.
authors.affiliation: Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Bharat G
authors.initials: BG
authors.lastname: Reddy
authors.affiliation: Dept. of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Qun
authors.initials: Q
authors.lastname: Dai
authors.affiliation: Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Carmel M
authors.initials: CM
authors.lastname: McNicholas
authors.affiliation: Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Fuller
authors.affiliation: Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233, United States.
authors.forename: John C
authors.initials: JC
authors.lastname: Kappes
authors.affiliation: Dept. of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address: duke2@uab.edu.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: DeLucas
mesh_terms: D000818:Animals; D053503:Epithelial Sodium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D053503:Epithelial Sodium Channels; D011994:Recombinant Proteins
keywords: ENaC; Epithelial sodium channel; GFP; GFP Affinity Column; Membrane Protein Expression; Membrane Protein Purification