Characterization of the E. coli glucose permease fused to the maltose-binding protein

• Published in: Journal of basic microbiology Vol. 48; no. 1; pp. 3 - 9
• Main Authors: Aboulwafa, Mohammad, Saier, Milton H. Jr
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.02.2008; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Blotting, Western; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Glucose - metabolism; IIGlc-MBP; Maltose-Binding Proteins; Methylglucosides - metabolism; Molecular Weight; Phosphoenolpyruvate - metabolism; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - genetics; Phosphoenolpyruvate Sugar Phosphotransferase System - isolation & purification; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphorylation; Protein Binding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Recombinant protein fusion technology
• ISSN: 0233-111X; 1521-4028
• DOI: 10.1002/jobm.200700263

The ptsG gene that encodes the major glucose transporter of Escherichia coli, IIGlc, was inserted into a pMALE-ampr expression vector down-stream of the malE gene which encodes the E. coli maltose-binding protein (MBP). IIGlc-MBP in the 2 h high speed supernatant of cell lysates eluted from a gel filtration column showing two activity peaks. The glucose-6-phosphate-dependent transphosphorylation (TP) activity of the membrane bound oligomeric peak 1 showed substrate inhibition while that of the soluble monomeric peak 2 did not. Purification of peak 2 yielded activity with weak substrate inhibition, and further gel filtration analyses showed that upon purification, some of the monomeric IIGlc-MBP associated to higher molecular size forms. Assays of the phosphoenolpyruvate-dependent and transphosphorylation reactions showed that the specific activity of the purified enzyme from peak 1 was approximately double that from peak 2. The results show that the monomeric IIGlc-MBP exhibits no substrate inhibition although the oligomeric form does. Purification promotes subunit association, an increase in catalytic activity, and restoration of substrate inhibition. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)