Abstract
Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Chromatography, High Pressure Liquid
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Enzyme Assays
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Escherichia coli / chemistry*
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Escherichia coli / enzymology*
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Escherichia coli / genetics
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Histidine / analogs & derivatives
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Histidine / chemistry
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Histidine / genetics
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Liver / chemistry
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Liver / cytology
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Mice
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Peroxisome-Targeting Signal 1 Receptor
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Receptors, Cytoplasmic and Nuclear / chemistry
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Receptors, Cytoplasmic and Nuclear / metabolism
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification*
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Temperature
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Ubiquitin-Activating Enzymes / biosynthesis
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Ubiquitin-Activating Enzymes / genetics
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Ubiquitin-Activating Enzymes / isolation & purification*
Substances
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Peroxisome-Targeting Signal 1 Receptor
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Receptors, Cytoplasmic and Nuclear
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Recombinant Fusion Proteins
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Histidine
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Ubiquitin-Activating Enzymes