title: Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.
abstract: Similar to the recombinant cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Sf9 insect cells, underglycosylated CFTR expressed in yeast is not effectively solubilized by a variety of commonly used detergents, requiring instead harsh alkali and SDS treatments, which would denature most proteins. Moreover, solubilized CFTR has a strong tendency to aggregate and form high-molecular-weight aggregates during subsequent purification. We report here that the mild detergent, lysophosphatidylglycerol (LPG), is a very effective detergent for solubilizing the CFTR expressed in both yeast and Sf9 insect cells. LPG solubilizes nearly 100% of the CFTR in yeast in the absence of NaCl and none in the presence of 1 M NaCl. It is also very potent in preventing aggregation of the CFTR during subsequent purification. Exploiting these characteristics, a rapid simple procedure for the purification of functional recombinant CFTR expressed in yeast has been developed. It includes selective CFTR solubilization in the presence and the absence of NaCl followed by nickel-chelate chromatography of His-tagged CFTR. The CFTR produced by this procedure is about 70% pure. Purified CFTR molecules were reconstituted into liposomes and then fused to planar lipid bilayers for single-channel recording. The reconstituted CFTR exhibits regulatory chloride channel activities with a slope conductance of 7.1 pS and a reversal potential of -32 mV. The effectiveness and simplicity of this new purification procedure for the CFTR should greatly facilitate a variety of biochemical and biophysical studies of this important protein. Furthermore, the potency of LPG in solubilizing the notoriously intractable underglycosylated CFTR suggest that this detergent may be useful for solubilizing the CFTR from other sources and for other difficult membrane proteins as well.
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
authors.forename: P
authors.initials: P
authors.lastname: Huang
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
authors.forename: G A
authors.initials: GA
authors.lastname: Scarborough
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000302:isolation & purification*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008246:Lysophospholipids; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D014162:Transfection
chemical_list: D003902:Detergents; D008246:Lysophospholipids; D011994:Recombinant Proteins; C026223:lysophosphatidylglycerol; D019005:Cystic Fibrosis Transmembrane Conductance Regulator