Human duodenal cytochrome b (Dcytb) is a transmembrane hemoprotein found in the duodenal brush border membrane and in erythrocytes. Dcytb has been linked to uptake of dietary iron and to ascorbate recycling in erythrocytes. Detailed biophysical and biochemical characterization of Dcytb has been limited by difficulties in expressing sufficient amounts of functional recombinant protein in yeast and insect cell systems. We have developed an Escherichia coil Rosetta-gami B(DE3) cell system for production of recombinant His-tagged human Dcytb with a yield of similar to 26 mg of purified, ascorbate-reducible cytochrome per liter of culture. The recombinant protein is readily solubilized with n-dodecyl-beta-D-maltoside and purified to electrophoretic homogeneity by one-step chromatography on cobalt affinity resin. The purified recombinant Dcytb has a heme to protein ratio very close to the theoretical value of 2 and retains functional reactivity with ascorbate, as assessed by spectroscopic and kinetic measurements. Ascorbate showed a marked kinetic selectivity for the high-potential heme center over the low-potential heme center in purified Dcytb. This new E. coli expression system for Dcytb offers similar to 7-fold improvement in yield and other substantial advantages over existing expression systems for reliable production of functional Dcytb at levels suitable for biochemical, biophysical and structural characterization. (C) 2011 Elsevier Inc. All rights reserved.