Use of Staphylococcus aureus 6-P-ß-Galactosidase and GFP as Fusion Partners for Lactose-Specific IIC Domain from Staphylococcus aureus

Claudia M. Kowolik and Wolfgang Hengstenberg

Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICB^lac) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-ßGalactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIC^lac domain with the IIB^lac domain of the lactose transporter. The activity of the 6-P-ß-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The V_max values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIC^lac domain and the complete lactose transporter (IICB^lac). The K_m values were only slightly increased indicating that the V_max values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the V_max value determined for the GFP-fused IIC^lac domain are higher than for the 6-P-ßGalactosidase-fused IIC^lac. These results suggest that the fusion with GFP enables a better interaction with the IIB^lac domain than the fusion with 6-Pß-Galactosidase. Moreover, the GFP-fused IIC^lac domain proved to be more stable than the 6-P-ß-Galactosidase fusion protein. ---

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