PMID:7737179

Citation	Peters, D, Frank, R and Hengstenberg, W (1995) 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. Eur. J. Biochem. 228:798-804
Abstract	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.
Links	PubMed
Keywords	Base Sequence; Cell Membrane/enzymology; Chromatography, Affinity/methods; Genetic Complementation Test; Histidine/chemistry; Kinetics; Metals; Molecular Sequence Data; Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics; Phosphoenolpyruvate Sugar Phosphotransferase System/isolation & purification; Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism; Phosphorylation; Staphylococcus aureus/enzymology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STRMU:PTLA		GO:0009401: phosphoenolpyruvate-dependent sugar phosphotransferase system	ECO:0000314:		P		Bacterial phosphotransferase-system-dependent phosphorylation of IIB-His was demonstrated in a photometric assay and by urea/polyacrylamide gel electrophoresis.	complete

References

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