THE GLUCOSE-TRANSPORTER OF ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION BY NI-CHELATE AFFINITY-CHROMATOGRAPHY OF THE IIBCGLC SUBUNIT()

The IIBC(Glc) transmembrane subunit of the glucose transporter of E. c oli containing a carboxy-terminal affinity tag consisting of six adjac ent 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 membran es was recovered. Adsorption to the nickel resin allows delipidation a s well as rapid detergent exchange. The procedure was used to demonstr ate exchange of subunits in the IIBC(Glc) dimer and it helds promise f or the investigation of other protein-protein interactions.