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Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.
Reddy, Bharat G; Dai, Qun; McNicholas, Carmel M; Fuller, Catherine M; Kappes, John C; DeLucas, Lawrence J.
Afiliación
  • Reddy BG; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
  • Dai Q; 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.
  • McNicholas CM; Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
  • Fuller CM; 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.
  • Kappes JC; 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, Un
  • DeLucas LJ; 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.
Protein Expr Purif ; 117: 67-75, 2016 Jan.
Article en En | MEDLINE | ID: mdl-26394093
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 αßγENaC, 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Expresión Génica / Canales Epiteliales de Sodio Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2016 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Expresión Génica / Canales Epiteliales de Sodio Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2016 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos