Lactose-Specific Enzyme II of the Phosphoenol Pyruvate-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

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The lactose-specific integral-membrane-protein enzyme II (IICBLac) of the bacterial phosphoenolpy-ruvate-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 non-denaturing conditions to homogenity. The purification procedure consits of a NaOH extraction step followed by solubilisation with Triton X-100, and metal-affinity chromatography using Ni2+-nitrilotriacetic acid resin. The purified recombinant His-tagged protein possessed subtrate specifity 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 phospho-transferase-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]-IICB1, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac-His in a phosphorylation assay.