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Biochim Biophys Acta. 2014 Nov;1838(11):2825-37. doi: 10.1016/j.bbamem.2014.07.016. Epub 2014 Jul 24.

A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR).

Author information

1
Department of Cell Biology and Biochemistry and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA.
2
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
3
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
4
Department of Medicine, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA.
5
Department of Optometry, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA.
6
Department of Medicine, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA; Department of Microbiology, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA; Department of Pathology, University of Alabama at Birmingham, 701 19th Street South, Birmingham, AL 35294-0007, USA; Birmingham Veterans Medical Center, Research Service, Birmingham, AL 35233, USA.
7
Department of Cell Biology and Biochemistry and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX 79430, USA. Electronic address: ina.urbatsch@ttuhsc.edu.

Abstract

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C12E8, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2-3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6-9days in MNG or Chaps, and 12-17days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.

KEYWORDS:

ABC transporter; ATP-binding cassette; Cystic fibrosis; Facial amphiphiles; Maltoside neopentyl glycol detergents

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