THE LACTOSE TRANSPORTER OF STAPHYLOCOCCUS-AUREUS - OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE HISTIDINE-TAGGED DOMAIN-IIC ANDDOMAIN-IIB

The lactose-specific enzyme II (IICBlac) of the phosphoenolpyruvate-de pendent phosphotransferase system (PTS) of Staphylococcus aureus coupl es translocation to phosphorylation of the transported lactose. It is composed of the N-teminal membrane-bound IIC domain, which includes th e sugar-binding site, and the C-terminal IIB domain, which contains th e 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 Esch erichia coli and purified by Ni2+ chelate affinity chromatography. 2 m g of IIClac-His(6) obtained from 10 g of cells and 12 mg of His(6)-IIB lac obtained from 8 g of wet cells were purified to homogeneity. 56% o f the total IIClac-His(6) activity present in the membranes could be r ecovered. Purification by affinity chromatography yields the opportuni ty to exchange the detergent. The K-m determined in an activity assay for IIClac-His(6) in the presence of the histidine-tagged IIBlac domai n (His(6)-IIBlac) was similar to the K-m determined for histidine-tagg ed IICBlac-His [Peters, D. & Hengstenberg, W. (1995) fur: J. Biochem. 228, 798-804], suggesting that substrate affinity is barely influenced by the expression of the domains as separate proteins. The V-max is r educed by a factor of 25 compared with IICBlac-His. His(6)-IIBlac also complements the activity of the IICBlac mutant C476S, which possesses an inactive IIB domain. This result indicates that IIC and IIB are fl exibly linked in such a way that free His(6)-IIBlac can displace the i nactive IIB domain from its contact site on the IIC domain. His(6)-IIB lac is shorter and more stable than a previously constructed IIB domai n (IIBlac-His) [Peters, D. & Hengstenberg, W. (1995) fur. J. Biochem. 228, 798-804)], which contained a C-terminal histidine tag. The K-m va lues for phosphoenolpyruvate-dependent phosphorylation of His(6)-IIBla c and IIBlac-His are nearly indistinguishable, suggesting that the loc ation of the affinity tag either at the N-terminal or at the C-termina l end of the domain does not influence the substrate affinity.