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Purification of the Tn 10 ‐specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein
Author(s) -
Aldema Mila L.,
McMurry Laura M.,
Walmsley Adrian R.,
Levy Stuart B.
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1996.359886.x
Subject(s) - biology , antiporter , fusion protein , biochemistry , microbiology and biotechnology , tetracycline , efflux , maltose binding protein , permease , protein a/g , vesicle associated membrane protein 8 , membrane protein , membrane , escherichia coli , recombinant dna , gene , antibiotics
Summary The bacterial tetracycline‐resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis‐tidine‐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 α‐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‐α‐deoxyte‐tracycline. These findings suggested that the purified protein was in a native state.