A string of enzymes, purification and characterization of a fusion protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli.
A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIAGlc, HPr, and enzyme I. The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni2+ chelate affinity chromatography. Approximately 3 mg of the fusion protein could be purified from 1 liter of culture. 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 HPr, 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. Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICBglc. Addition of extra IICBGlc subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIAGlc subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect. An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties.
Mao
Q
Q
Institute of Biochemistry, University of Berne, Switzerland.
Schunk
T
T
Gerber
B
B
Erni
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008055:Lipids / Q000494:pharmacology; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D008055:Lipids; D011993:Recombinant Fusion Proteins; D017830:Octoxynol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C031629:phosphoenolpyruvate-glucose phosphotransferase; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase