A STRING OF ENZYMES, PURIFICATION AND CHARACTERIZATION OF A FUSION PROTEIN COMPRISING THE 4 SUBUNITS OF THE GLUCOSE PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI

A multidomain protein comprising the four subunits of the glucose phos photransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIA(Glc), HPr, and enzyme I, The subunits were linked in the above or der with Ala-Pro rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni2+ chelate affinity chromatography, Approxi mately 3 mg of the fusion protein could be purified from 1 liter of cu lture, The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits, The mannose transporter, which also requires enzyme I and H Pr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed, Tran sphosphorylation activity of the fusion protein was almost indistingui shable from the wild-type IICBGlc. Addition of extra IICBGlc subunit c ould significantly stimulate the phosphotransferase activity of the fu sion protein, addition of extra IIA(Glc) subunit and enzyme I, in cont rast, was slightly inhibitory, and HPr had almost no effect, An optima l detergent-lipid ratio is required for maximum activity of the fusion protein, Our results suggest that Ala-Pro-rich linker sequences may b e of general use for the construction of catalytically active fusion p roteins with novel properties.