Lactose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Purification of the histidine-tagged transmembrane component IICBLac and its hydrophilic IIB domain by metal-affinity chromatography, and functional characterization.
The lactose-specific integral-membrane-protein enzyme II (IICBLac) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus catalyses the uptake and phosphorylation of lactose. It consists of an N-terminal membrane-spanning IIC domain and a C-terminal hydrophilic IIB domain. IICBLac was fused with a C-terminal tag of six histidine residues using recombinant DNA technology. The resulting protein, IICBLac-His, was produced in Escherichia coli and purified under nondenaturing conditions to homogenity. The purification procedure consists of a NaOH extraction step followed by solubilisation with Triton X-100, and metal-affinity chromatography using Ni(2+)-nitrilotriacetic acid resin. The purified recombinant His-tagged protein possessed substrate specificity identical to that of the wild-type protein. To investigate the hydrophilic IIB domain, the DNA sequence coding for IIB and the His tag were fused in-frame to a DNA sequence specific for an initiation signal. The overproduced recombinant IIBLac-His was obtained by metal-affinity chromatography in pure form. Bacterial phosphotransferase-system-dependent phosphorylation of IIB-His was demonstrated in a photometric assay and by urea/polyacrylamide gel electrophoresis. The phosphorylation activity of the mutant protein [C476S]-IICBLac, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac-His in a phosphorylation assay.
Peters
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D
Abteilung für Biologie, Ruhr-Universität Bochum, Germany.
Frank
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R
Hengstenberg
W
W
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D005816:Genetic Complementation Test; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D013211:Staphylococcus aureus / Q000201:enzymology*
D008670:Metals; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C029022:phosphoenolpyruvate-lactose dependent phosphotransferase system