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Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator
- Source :
- Protein expression and purification. 25(1)
- Publication Year :
- 2002
-
Abstract
- 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.
- Subjects :
- DNA, Complementary
Protein Conformation
Recombinant Fusion Proteins
Detergents
Molecular Sequence Data
Cystic Fibrosis Transmembrane Conductance Regulator
medicine.disease_cause
Cleavage (embryo)
Thioredoxins
Affinity chromatography
medicine
Escherichia coli
Amino Acid Sequence
Chromatography, High Pressure Liquid
Micelles
biology
Sequence Homology, Amino Acid
Circular Dichroism
Cell Membrane
Membrane Proteins
Fusion protein
Cystic fibrosis transmembrane conductance regulator
Protein Structure, Tertiary
Membrane
Biochemistry
Membrane protein
biology.protein
Electrophoresis, Polyacrylamide Gel
Thioredoxin
Biotechnology
Subjects
Details
- ISSN :
- 10465928
- Volume :
- 25
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Protein expression and purification
- Accession number :
- edsair.doi.dedup.....7c9e717b0759a4f310f8aa72d66bf8f0