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. 2012 Jul;51(3):254-61.
doi: 10.1007/s12033-011-9463-x.

High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1

Andreia F Carvalho  1 , Manuel P Pinto, Cláudia P Grou, Rui Vitorino, Pedro Domingues, Fumiaki Yamao, Clara Sá-Miranda, Jorge E Azevedo
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High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1

Andreia F Carvalho et al. Mol Biotechnol. 2012 Jul.

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.

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