High-Yield Expression in Escherichia coli and Purification of Mouse Ubiquitin-Activating Enzyme E1
High-Yield Expression in Escherichia coli and Purification of Mouse Ubiquitin-Activating Enzyme E1
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DOI：
10.1007/s12033-011-9463-x
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发表时间：
2012-07-01
影响因子：
2.6
通讯作者：
Azevedo, Jorge E.
中科院分区：
文献类型：
--
作者：
Carvalho, Andreia F.;Pinto, Manuel P.;Azevedo, Jorge E.
泛素领域的研究需要大量的泛素激活酶（E1）用于体外泛素化测定。通常，哺乳动物酶从天然来源分离或使用杆状病毒/昆虫细胞蛋白表达系统重组产生。大肠杆菌很少用于生产哺乳动物E1，可能是由于这种高分子量蛋白的不稳定性和不溶性。在这份报告中，我们表明，5-10毫克的组氨酸标记的小鼠E1可以很容易地从一个11 E。大肠杆菌培养。发现蛋白质诱导步骤期间的低温对于获得活性酶至关重要。
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.