Purification of the Tn10-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.
Aldema
M L
ML
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
McMurry
L M
LM
Walmsley
A R
AR
Levy
S B
SB
D017920:Antiporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013753:Tetracycline Resistance / Q000235:genetics*; D013754:Tetracyclines / Q000378:metabolism*
D017920:Antiporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D003902:Detergents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013754:Tetracyclines; C079051:tetA protein, Bacteria; C033223:polyhistidine; D007544:Isopropyl Thiogalactoside; D006639:Histidine