期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 383, 期 2, 页码 281-287出版社
ELSEVIER SCIENCE INC
DOI: 10.1006/abbi.2000.2089
关键词
sulfite oxidase; molybdenum; molybdopterin; mob; molybdenum domain; tungsten
资金
- NIGMS NIH HHS [GM44283] Funding Source: Medline
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. (C) 2000 Academic Press.
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