Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH : ubiquinone oxidoreductase (Complex I)

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichi a coli is composed of 13 different subunits. The corresponding genes are or ganized in the nuo-operon (from NADH: ubiquinone oxidoreductase) at min 51 of the E. coli chromosome, To study the structure and function of this comp lex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promot or region with the phage-T7 RNA polymerase promotor and by expressing the g enes with the T7 RNA polymerase coded on an inducible plasmid, It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membrane s that complex I is overproduced 4-fold after induction. Complex I was isol ated by chromatographic steps performed in the presence of dodecyl maltosid e. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability o ver a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.