Purification of UhpT, the Sugar Phosphate Transporter ofEscherichia coli

Abstract

To purify UhpT, the sugar phosphate carrier ofEscherichia coli,we constructed a variant (HisUhpT) in which 10 tandem histidine residues were placed at the UhpT N terminus and then used Ni2+–agarose affinity chromatography of detergent-solubilized proteins. Membrane vesicles from a strain overexpressing HisUhpT were extracted at pH 7.4 with either 1.5%n-octyl-β-d-glucopyranoside (octylglucoside) or 1.5%n-dodecyl-β-d-maltoside (dodecylmaltoside) in 200 mmsodium chloride, 100 mmpotassium phosphate, 50 mmglucose 6-phosphate, 10–20% glycerol, 0.2%E. coliphospholipid, and 5 mmβ-mercaptoethanol. After the detergent extract was applied to a Ni2+–agarose column, nonspecifically bound material was removed by washing at pH 7 with the same buffer also containing 50 mmimidazole. Purified HisUhpT was released subsequently, when sodium chloride was replaced with 300 mmimidazole or 100 mmEDTA, giving an overall yield of about 25 μg HisUhpT/mg vesicle protein. Whether eluted by imidazole or EDTA in either octylglucoside or dodecylmaltoside, purified HisUhpT showed a specific activity of 2.5–3 μmol/min per milligram of protein as monitored by [14C]glucose 6-phosphate transport by proteoliposomes loaded with 100 mmpotassium phosphate. This corresponded to a calculated turnover number near 20 s−1for the heterologous exchange of external sugar phosphate with internal phosphate. At low temperature (4°C) HisUhpT retained full activity in either octylglucoside or dodecylmaltoside; however, at elevated temperature (≥23°C), the protein displayed a marked lability in octylglucoside (t12 = 11 min), but not in dodecylmaltoside (t12 ≥ 200–300 min).