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

作者：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^*

来源：Biochimica et Biophysica Acta-Biomembranes, 2014, 1838(11): 2825-2837.

DOI：10.1016/j.bbamem.2014.07.016

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

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/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 allcylmaltosides, 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 CETR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/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 6-9 days in MNG or Chaps, and 12-17 days 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.

• 出版日期2014-11