The glucose transporter of Escherichia coli. Purification and characterization by Ni+ chelate affinity chromatography of the IIBCGlc subunit.
The IIBCGlc transmembrane subunit of the glucose transporter of E. coli containing a carboxy-terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography. The protein was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% of the phosphotransferase activity present in cell membranes was recovered. Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange of subunits in the IIBCGlc dimer and it holds promise for the investigation of other protein-protein interactions.
Waeber
U
U
Institute for Biochemistry, University of Bern, Switzerland.
Buhr
A
A
Schunk
T
T
Erni
B
B
D001483:Base Sequence; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009532:Nickel; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D015183:Restriction Mapping
D002614:Chelating Agents; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D009532:Nickel