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 tetrac yclines. The tetA gene was cloned behind a T7 promoter/lac operator in a plasmid that provided fusion of TetA to a polyhistidine-carboxy ter minal tail. A second plasmid provided a regulated T7 RNA polymerase. T he specific activity of the TetA fusion protein was between 10-40% tha t of the wild-type protein as assayed by tetracycline resistance in ce lls and by transport in membrane vesicles. The fusion protein, overpro duced approximately 3-13-fold, was purified by nickel chelation chroma tography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an alpha-helix content o f 54-64%, close to the 68% predicted from the amino acid sequence by h ydropathy analysis (12 membrane-spanning helices) for the native prote in in the membrane bilayer. Fluorescence studies showed binding activi ty of the purified protein to its substrate, the tetracycline analogue lopentylthio)-5-hydroxy-6-alpha-deoxytetracycline. These findings sug gested that the purified protein was in a native state.