The lactose transporter of Staphylococcus aureus--overexpression, purification and characterization of the histidine-tagged domains IIC and IIB.
The lactose-specific enzyme II (IICBlac) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of Staphylococcus aureus couples translocation to phosphorylation of the transported lactose. It is composed of the N-terminal membrane-bound IIC domain, which includes the sugar-binding site, and the C-terminal IIB domain, which contains the phosphorylation site at Cys476. IIC (residues 1-461) fused with a C-terminal affinity tag of six histidine residues and IIB (residues 461-570) fused with an N-terminal histidine tag were overexpressed in Escherichia coli and purified by Ni2+ chelate affinity chromatography. 2 mg of IIClac-His6 obtained from 10 g of cells and 12 mg of His6-IIBlac obtained from 8 g of wet cells were purified to homogeneity. 56% of the total IIClac-His6 activity present in the membranes could be recovered. Purification by affinity chromatography yields the opportunity to exchange the detergent. The Km determined in an activity assay for IIClac-His6 in the presence of the histidine-tagged IIBlac domain (His6-IIBlac) was similar to the Km determined for histidine-tagged IICBlac-His [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804], suggesting that substrate affinity is barely influenced by the expression of the domains as separate proteins. The Vmax is reduced by a factor of 25 compared with IICBlac-His. His6-IIBlac also complements the activity of the IICBlac mutant C476S, which possesses an inactive IIB domain. This result indicates that IIC and IIB are flexibly linked in such a way that free His6-IIBlac can displace the inactive IIB domain fron its contact site on the IIC domain. His6-IIBlac is shorter and more stable than a previously constructed IIB domain (IIBlac-His) [Peters, D. & Hengstenberg, W. (1995) Eur. J. Biochem. 228, 798-804)], which contained a C-terminal histidine tag. The Km values for phosphoenolpyruvate-dependent phosphorylation of His6-IIBlac and IIBlac-His are nearly indistinguishable, suggesting that the location of the affinity tag either at the N-terminal or at the C-terminal end of the domain does not influence the substrate affinity.
Kowolik
C M
CM
Department of Biology, University of Bochum, Germany.
Hengstenberg
W
W
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology*; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; D006639:Histidine; C021499:lactose permease