title: Purification of the cystic fibrosis transmembrane conductance regulator protein expressed in Saccharomyces cerevisiae.
abstract: Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal. We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments. In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.
authors.affiliation: Faculty of Life Sciences, University of Manchester.
authors.forename: Naomi
authors.initials: N
authors.lastname: Pollock
authors.affiliation: Faculty of Life Sciences, University of Manchester.
authors.forename: Natasha
authors.initials: N
authors.lastname: Cant
authors.affiliation: Faculty of Life Sciences, University of Manchester.
authors.forename: Tracy
authors.initials: T
authors.lastname: Rimington
authors.affiliation: Faculty of Life Sciences, University of Manchester; bob.ford@manchester.ac.uk.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
mesh_terms: D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D005960:Glucosides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005960:Glucosides; D010743:Phospholipids; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C040358:dodecyl maltoside