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Reconstitution of the metal-tetracycline/H+antiporter ofEscherichia coliin proteoliposomes including F0F1-ATPase

Authors
Journal
FEBS Letters
0014-5793
Publisher
Wiley Blackwell (John Wiley & Sons)
Publication Date
Volume
374
Issue
1
Identifiers
DOI: 10.1016/0014-5793(95)01079-t
Keywords
  • Tetracycline
  • Antiporter
  • Tetracycline/H+Antiporter
  • H+-Atpase
  • Reconstitution
  • Proteoliposome
Disciplines
  • Biology

Abstract

Abstract The tetracycline resistance gene ( tetA) was cloned downstream of the lac promoter. When expression of the tetA gene in E. coli cells carrying the lac I q gene was induced with isopropyl β- d-thiogalactopyranoside, the tetracycline resistance protein (TetA) was overproduced, amounting to about 30% of the integral cytoplasmic membrane protein. Essentially pure TetA protein could be obtained by solubilization with 1.25% n- octyl-β- d-glucopyranoside and one-step purification by DEAE Sepharose CL-6B column chromatography. The TetA protein was incorporated into proteoliposomes with F 0F 1-ATPase. The proteoliposomes exhibited [ 3H]tetracycline transport dependent on ATP hydrolysis. The specific activity was about 2 nmol/mg protein/min. The proteoliposomes also showed H + efflux coupled with tetracycline influx. Tetracycline/H + antiport by proteoliposomes reconstituted with the Ser-65 → Cys mutant TetA protein was inhibited by N-ethylmaleimide. These results proved for the first time that the tetracycline/H + antiport is only mediated by the TetA protein.

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