title: High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system.
abstract: 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 coli Rosetta-gami B(DE3) cell system for production of recombinant His-tagged human Dcytb with a yield of ∼26 mg of purified, ascorbate-reducible cytochrome per liter of culture. The recombinant protein is readily solubilized with n-dodecyl-β-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 ∼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.
authors.affiliation: Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030, United States. Wen.Liu.2@uth.tmc.edu
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: Ah-Lim
authors.initials: AL
authors.lastname: Tsai
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kulmacz
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D011994:Recombinant Proteins; D010088:Oxidoreductases; C422926:CYBRD1 protein, human; D001205:Ascorbic Acid