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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Abbreviations
- DTT:
-
Dithiothreitol
- E1:
-
Ubiquitin-activating enzyme
- E2:
-
Ubiquitin-conjugating enzyme
- E3:
-
Ubiquitin ligase
- GST-Ub:
-
Glutathione S-transferase-tagged ubiquitin
- IPTG:
-
Isopropyl 1-thio-β-d-galactopyranoside
- PAGE:
-
Polyacrylamide gel electrophoresis
- UbcH5c and UbcH7:
-
Human ubiquitin-conjugating enzyme 5c and 7, respectively
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Acknowledgments
We thank Dr. Martin Scheffner, University of Konstanz, Germany, for the kind gift of pET3a-UbcH7 plasmid. This study was supported by Fundação para a Ciência e Tecnologia (COMPETE program), and Fundo Europeu de Desenvolvimento Regional, Portugal (grants PTDC/BIA-BCM/64771/2006, PEst-C/QUI/UI0062/2011 and PEst-C/SAU/LA0002/2011). A.F.C. is supported by Programa Ciência—funded by POPH-QREN-Tipologia 4.2-Promoção do Emprego Científico, by Fundo Social Europeu and by national funds from MCTES. M.P.P. and C.P.G. are supported by Fundação para a Ciência e Tecnologia.
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Carvalho, A.F., Pinto, M.P., Grou, C.P. et al. High-Yield Expression in Escherichia coli and Purification of Mouse Ubiquitin-Activating Enzyme E1. Mol Biotechnol 51, 254–261 (2012). https://doi.org/10.1007/s12033-011-9463-x
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DOI: https://doi.org/10.1007/s12033-011-9463-x