Purification of UhpT, the sugar phosphate transporter of Escherichia coli.
To purify UhpT, the sugar phosphate carrier of Escherichia coli, we constructed a variant (HisUhpT) in which 10 tandem histidine residues were placed at the UhpT N terminus and then used Ni(2+)-agarose affinity chromatography of detergent-solubilized proteins. Membrane vesicles from a strain overexpressing His-UhpT were extracted at pH 7.4 with either 1.5% n-octyl-beta-D-glucopyranoside (octylglucoside) or 1.5% n-dodecyl-beta-D-maltoside (dodecylmaltoside) in 200 mM sodium chloride, 100 mM potassium phosphate, 50 mM glucose 6-phosphate, 10-20% glycerol, 0.2% E. coli phospholipid, and 5 mM beta-mercaptoethanol. After the detergent extract was applied to a Ni(2+)-agarose column, nonspecifically bound material was removed by washing at pH 7 with the same buffer also containing 50 mM imidazole. Purified HisUhpT was released subsequently, when sodium chloride was replaced with 300 mM imidazole or 100 mM EDTA, giving an overall yield of about 25 micrograms 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 mumol/min per milligram of protein as monitored by [14C]glucose 6-phosphate transport by proteoliposomes loaded with 100 mM potassium phosphate. This corresponded to a calculated turnover number near 20 s-1 for the heterologous exchange of external sugar phosphate with internal phosphate. At low temperature (4 degrees C) HisUhpT retained full activity in either octylglucoside or dodecylmaltoside; however, at elevated temperature (> or = 23 degrees C), the protein displayed a marked lability in octylglucoside (t1/2 = 11 min), but not in dodecylmaltoside (t1/2 > or = 200-300 min).
Tamai
E
E
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Fann
M C
MC
Tsuchiya
T
T
Maloney
P C
PC
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003902:Detergents; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005960:Glucosides; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013403:Sugar Phosphates / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D013403:Sugar Phosphates; C063498:UhpT protein, E coli; D006639:Histidine; C040358:dodecyl maltoside; D009532:Nickel