LACTOSE-SPECIFIC ENZYME-II OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF STAPHYLOCOCCUS-AUREUS - PURIFICATION OF THE HISTIDINE-TAGGED TRANSMEMBRANE COMPONENT IICBL(LAC) 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 l actose. It consists of an N-terminal membrane-spanning IIC domain and a C-terminal hydrophilic IIB domain. IICBLac was fused with a C-termin al 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 purificatio n procedure consits of a NaOH extraction step followed by solubilisati on with Triton X-100, and metal-affinity chromatography using Ni2+-nit rilotriacetic acid resin. The purified recombinant His-tagged protein possessed subtrate specifity identical to that of the wild-type protei n. 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 pho sphotransferase-system-dependent phosphorylation of IIB-His was demons trated in a photometric assay and by urea/polyacrylamide gel electroph oresis. The phosphorylation activity of the mutant protein [C476S] IIC BLac, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac-His in a phosphorylation assay.