Reconstitution of the metal‐tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1‐ATPase

[摘要]

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% and one-step purification by DEAE Sepharose CL-6B column chromatography. The TetA protein was incorporated into proteoliposomes with F₀F₁-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.

[学科分类] 生物化学/生物物理

[关键词] Tetracycline;Antiporter;Tetracycline/H+ antiporter;H+-ATPase;Reconstitution;Proteoliposome;TetA;tetracycline resistance protein;MOPS;3-(N-morpholino)propanesulfonic acid;MES;2-(N-morpholino)ethanesulfonic acid;tricine;N-tris(hydroxymethyl)methylglycine;octylglucoside;NEM;N-ethylmaleimide;TC;tetracycline