THE GLUCOSE-TRANSPORTER OF ESCHERICHIA-COLI - OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF FUNCTIONAL DOMAINS

The glucose transporter of the bacterial phosphotransferase system cou ples vectorial translocation to phosphorylation of the transported sug ar. It consists of a transmembrane subunit (IICE(Glc)) and a hydrophil ic subunit (IIA(Glc)). The IICBGlc subunit consists of two domains. Th e NH2-terminal IIC domain (residues 1-386) spans the membrane eight ti mes and contains the substrate binding site. The COOH-terminal hydroph ilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys(421)) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIA(Glc) subunit to the transported solute. Starting f rom a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coil, and purified by Ni2+ chelate affinity chromatogra phy. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be p urified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc -6H separately have a three times longer generation time on glucose mi nimal medium than cells expressing wild-type IICBGlc. The rate of IIBG lc-6H phosphorylation determined in a nitrocellulose filter binding as say is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of th e control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are f lexibly linked such that a free IIB domain can displace an inactive II B domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by is otope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Ge mmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Bioche m. 219, 945-952).