Skip to main content
Log in

High-Yield Expression in Escherichia coli and Purification of Mouse Ubiquitin-Activating Enzyme E1

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

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

References

  1. Schwartz, A. L., & Ciechanover, A. (2009). Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annual Review of Pharmacology and Toxicology, 49, 73–96.

    Article  CAS  Google Scholar 

  2. Pickart, C. M., & Eddins, M. J. (2004). Ubiquitin: Structures, functions, mechanisms. Biochimica et Biophysica Acta, 1695, 55–72.

    Article  CAS  Google Scholar 

  3. Pierce, N. W., Kleiger, G., Shan, S. O., & Deshaies, R. J. (2009). Detection of sequential polyubiquitylation on a millisecond timescale. Nature, 462, 615–619.

    Article  CAS  Google Scholar 

  4. Ciechanover, A., Heller, H., Katz-Etzion, R., & Hershko, A. (1981). Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system. Proceedings of the National Academy of Sciences of the United States of America, 78, 761–765.

    Article  CAS  Google Scholar 

  5. Haas, A. L., Warms, J. V., Hershko, A., & Rose, I. A. (1982). Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. The Journal of Biological Chemistry, 257, 2543–2548.

    CAS  Google Scholar 

  6. Ciechanover, A., Elias, S., Heller, H., & Hershko, A. (1982). “Covalent affinity” purification of ubiquitin-activating enzyme. The Journal of Biological Chemistry, 257, 2537–2542.

    CAS  Google Scholar 

  7. Haas, A. L. (2005). Purification of E1 and E1-like enzymes. Methods in Molecular Biology, 301, 23–35.

    CAS  Google Scholar 

  8. Beaudenon, S., & Huibregtse, J. M. (2005). High-level expression and purification of recombinant E1 enzyme. Methods in Enzymology, 398, 3–8.

    Article  CAS  Google Scholar 

  9. Carlile, C. M., Pickart, C. M., Matunis, M. J., & Cohen, R. E. (2009). Synthesis of free and proliferating cell nuclear antigen-bound polyubiquitin chains by the RING E3 ubiquitin ligase Rad5. The Journal of Biological Chemistry, 284, 29326–29334.

    Article  CAS  Google Scholar 

  10. Everett, R. D., Boutell, C., McNair, C., Grant, L., & Orr, A. (2010). Comparison of the biological and biochemical activities of several members of the alphaherpesvirus ICP0 family of proteins. Journal of Virology, 84, 3476–34787.

    Article  CAS  Google Scholar 

  11. Jin, L., Williamson, A., Banerjee, S., Philipp, I., & Rape, M. (2008). Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell, 133, 653–665.

    Article  CAS  Google Scholar 

  12. Nagai, Y., Kaneda, S., Nomura, K., Yasuda, H., Seno, T., & Yamao, F. (1995). Ubiquitin-activating enzyme, E1, is phosphorylated in mammalian cells by the protein kinase Cdc2. Journal of Cell Science, 108, 2145–2152.

    CAS  Google Scholar 

  13. Nakagawa, S., & Huibregtse, J. M. (2000). Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Molecular and Cellular Biology, 20, 8244–8253.

    Article  CAS  Google Scholar 

  14. Sun, B., Jeyaseelan, K., Chung, M. C., & Teo, T. S. (1997). Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression. Gene, 196, 19–23.

    Article  CAS  Google Scholar 

  15. Tokgoz, Z., Bohnsack, R. N., & Haas, A. L. (2006). Pleiotropic effects of ATP·Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme. The Journal of Biological Chemistry, 281, 14729–14737.

    Article  Google Scholar 

  16. Zheng, M., Liu, J., Yang, Z., Gu, X., Li, F., Lou, T., et al. (2010). Expression, purification and characterization of human ubiquitin-activating enzyme, UBE1. Molecular Biology Reports, 37, 1413–1419.

    Article  CAS  Google Scholar 

  17. Imai, N., Kaneda, S., Nagai, Y., Seno, T., Ayusawa, D., Hanaoka, F., et al. (1992). Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1. Gene, 118, 279–282.

    Article  CAS  Google Scholar 

  18. Nuber, U., Schwarz, S., Kaiser, P., Schneider, R., & Scheffner, M. (1996). Cloning of human ubiquitin-conjugating enzymes UbcH6 and UbcH7 (E2–F1) and characterization of their interaction with E6-AP and RSP5. The Journal of Biological Chemistry, 271, 2795–2800.

    Article  CAS  Google Scholar 

  19. Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W., & Klevit, R. E. (2006). A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Molecular Cell, 21, 873–880.

    Article  CAS  Google Scholar 

  20. Ferro, A., Carvalho, A. L., Teixeira-Castro, A., Almeida, C., Tome, R. J., Cortes, L., et al. (2007). NEDD8: A new ataxin-3 interactor. Biochimica et Biophysica Acta, 1773, 1619–1627.

    Article  CAS  Google Scholar 

  21. Grou, C. P., Carvalho, A. F., Pinto, M. P., Huybrechts, S. J., Sa-Miranda, C., Fransen, M., et al. (2009). Properties of the ubiquitin-pex5p thiol ester conjugate. The Journal of Biological Chemistry, 284, 10504–10513.

    Article  CAS  Google Scholar 

  22. Grou, C. P., Carvalho, A. F., Pinto, M. P., Wiese, S., Piechura, H., Meyer, H. E., et al. (2008). Members of the E2D (UbcH5) family mediate the ubiquitination of the conserved cysteine of Pex5p, the peroxisomal import receptor. The Journal of Biological Chemistry, 283, 14190–14197.

    Article  CAS  Google Scholar 

  23. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  24. Grou, C. P., Carvalho, A. F., Pinto, M. P., Alencastre, I. S., Rodrigues, T. A., Freitas, M. O., et al. (2009). The peroxisomal protein import machinery-a case report of transient ubiquitination with a new flavor. Cellular and Molecular Life Sciences, 66, 254–262.

    Article  CAS  Google Scholar 

  25. Lanyon-Hogg, T., Warriner, S. L., & Baker, A. (2010). Getting a camel through the eye of a needle: The import of folded proteins by peroxisomes. Biology of the Cell, 102, 245–263.

    Article  CAS  Google Scholar 

  26. Carvalho, A. F., Pinto, M. P., Grou, C. P., Alencastre, I. S., Fransen, M., Sa-Miranda, C., et al. (2007). Ubiquitination of mammalian Pex5p, the peroxisomal import receptor. The Journal of Biological Chemistry, 282, 31267–31272.

    Article  CAS  Google Scholar 

  27. Pelzer, C., Kassner, I., Matentzoglu, K., Singh, R. K., Wollscheid, H. P., Scheffner, M., et al. (2007). UBE1L2, a novel E1 enzyme specific for ubiquitin. The Journal of Biological Chemistry, 282, 23010–23014.

    Article  CAS  Google Scholar 

  28. Webster, J. M., Tiwari, S., Weissman, A. M., & Wojcikiewicz, R. J. (2003). Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+. The Journal of Biological Chemistry, 278, 38238–38246.

    Article  CAS  Google Scholar 

  29. Dodt, G., Braverman, N., Wong, C., Moser, A., Moser, H. W., Watkins, P., et al. (1995). Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nature Genetics, 9, 115–125.

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jorge E. Azevedo.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-011-9463-x

Keywords

Navigation