Purification of the Tn 10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein

The bacterial tetracycline-resistance determinant from Tn10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/lac operator in a plasmid that provided fusion of TetA to a polyhistidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10-40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3-13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an alpha-helix content of 54-64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-alpha-deoxytetracycline. These findings suggested that the purified protein was in a native state.