Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator.
We describe a rapid method for the expression and purification of two hydrophobic protein constructs derived from the membrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein associated with cystic fibrosis. The proteins have no sequence homology but are both predicted to contain two membrane-spanning segments. The protocol involves the expression of CFTR constructs as thioredoxin fusion proteins in Escherichia coli, followed by partial purification by affinity chromatography, removal of the thioredoxin moiety by proteolytic cleavage in the presence of detergent, and final purification by reversed-phase high-performance liquid chromatography. The method yields milligram amounts of purified constructs that spontaneously insert into detergent micelles in alpha-helical conformation. We predict that this protocol will be applicable to a variety of proteins of similar size and hydrophobicity.
Therien
Alex G
AG
Division of Structural Biology and Biochemistry, Research Institute, Toronto, Ontario, Canada.
Glibowicka
Mira
M
Deber
Charles M
CM
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008823:Micelles; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000378:metabolism
D018076:DNA, Complementary; D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013879:Thioredoxins