title: Optimization of expression of human sulfite oxidase and its molybdenum domain.
abstract: The conditions for the heterologous expression of both untagged and His-tagged human sulfite oxidase in Escherichia coli have been optimized. Maximum production of active enzyme requires expression in a mob- cell strain at low levels of the inducer. Using these conditions, 3.9-5.6 mg of untagged and 15 mg of His-tagged sulfite oxidase were isolated per liter of cell culture. These represent significantly higher levels than previously reported for any molybdopterin-containing protein. High levels of enzyme activity and molybdenum incorporation were maintained despite the increase in yield, and no significant differences in kinetic properties were observed between the tagged and untagged sulfite oxidase. Additionally, the molybdenum domain of sulfite oxidase was expressed in a stable, active form as a His-tagged protein. The molybdenum domain was also expressed in the presence of tungstate to enable examination of the molybdopterin-tungsten form of sulfite oxidase.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Temple
authors.forename: T N
authors.initials: TN
authors.lastname: Graf
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
mesh_terms: D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000096:biosynthesis* / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D014414:Tungsten / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D003574:Cytochrome c Group; D005292:Ferricyanides; D011994:Recombinant Proteins; C007931:hexacyanoferrate III; D008982:Molybdenum; D050862:Oxidoreductases Acting on Sulfur Group Donors; D014414:Tungsten