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

• Published in: Biochimica et biophysica acta 2014-11, Vol.1838 (11), p.2825-2837
• Main Authors: Hildebrandt, Ellen, Zhang, Qinghai, Cant, Natasha, Ding, Haitao, Dai, Qun, Peng, Lingling, Fu, Yu, DeLucas, Lawrence J., Ford, Robert, Kappes, John C., Urbatsch, Ina L.
• Format: Article
• Language: English
• Subjects: ABC transporter; ATP-binding cassette; Cystic fibrosis; Facial amphiphiles; Maltoside neopentyl glycol detergents
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2014.07.016

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. [Display omitted] •Detergents were screened for human CFTR purification, function and stability.•Purified CFTR hydrolyzes ATP without detergent removal if supplemented with lipid.•CFTR is monodisperse and can be concentrated above 1mg/ml without aggregation.•Activity and stability are greatest in neopentyl maltoside or facial amphiphiles.