PURIFICATION OF UHPT, THE SUGAR-PHOSPHATE TRANSPORTER OF ESCHERICHIA-COLI

To purify UhpT, the sugar phosphate carrier of Escherichia coli, we co nstructed a variant (HisUhpT) in which 10 tandem histidine residues we re placed at the UhpT N terminus and then used Ni2+-agarose affinity c hromatography of detergent-solubilized proteins. Membrane vesicles fro m a strain overexpressing HisUhpT were extracted at pH 7.4 with either 1.5% n-octyl-beta-D-glucopyranoside (octylglucoside) or 1.5% n-dodecy l-beta-D-maltoside (dodecylmaltoside) in 200 mM sodium chloride, 100 m M potassium phosphate, 50 nM glucose 6-phosphate, 10-20% glycerol, 0.2 % E. coli phospholipid, and 5 mM beta-mercaptoethanol, After the deter gent extract was applied to a Ni2+-agarose column, nonspecifically bou nd material was removed by washing at pH 7 with the same buffer also c ontaining 50 mM imidazole. Purified HisUhpT was released subsequently, when sodium chloride was replaced with 300 mM imidazole or 100 mM EDT A, giving an overall yield of about 25 mu g HisUhpT/mg vesicle protein . Whether eluted by imidazole or EDTA in either octylglucoside or dode cylmaltoside, purified HisUhpT showed a specific activity of 2.5-3 mu mol/min per milligram of protein as monitored by [C-14]glucose 6-phosp hate transport by proteoliposomes loaded with 100 mM potassium phospha te, This corresponded to a calculated turnover number near 20 s(-1) fo r the heterologous exchange of external sugar phosphate with internal phosphate, At low temperature (4 degrees C) HisUhpT retained full acti vity in either octylglucoside or dodecylmaltoside; however, at elevate d temperature (greater than or equal to 23 degrees C), the protein dis played a marked lability in octylglucoside (t 1/2 = 11 min), but not i n dodecylmaltoside (t 1/2 greater than or equal to 200-300 min). (C) 1 997 Academic Press.