Purification of uhpt, the sugar phosphate transporter of Escherichia coli

Eiji Tamai, Mon Chou Fann, Tomofusa Tsuchiya, Peter C. Maloney

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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 Ni2+-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-β-D-glucopyranoside (octylglucoside) or 1.5% n-dodecyl-β-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 β-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 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 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 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°C) HisUhpT retained full activity in either octylglucoside or dodecylmaltoside; however, at elevated temperature (≤23°C), the protein displayed a marked lability in octylglucoside (t 1/4 = 11 min), but not in dodecylmaltoside (t 1/4 ≤ 200-300 min).

Original languageEnglish (US)
Pages (from-to)275-282
Number of pages8
JournalProtein Expression and Purification
Volume10
Issue number2
DOIs
StatePublished - Jul 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology

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