Abstract
We previously described the establishment of a binary vector system that allows co-expression of SUMO conjugation enzymes and a target protein of interest, leading to efficient SUMO modification and the production of a large amount of recombinant SUMO-modified proteins in Escherichia coli. The advantages of this E. coli expression/modification approach include scalability of experiments, low cost, fast growth, and a lack of proteases that cleave the isopeptide linkage between SUMO and the target protein. Thus, this E. coli method provides a useful alternative to authentic SUMO modification assays, such as in vitro SUMO conjugation and in vivo SUMO modification using baculovirus or mammalian cell culture, that are usually complicated, time-consuming and expensive.
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References
Uchimura, Y., Nakamura, M., Sugasawa, K., Nakao, M., and Saitoh, H. (2004) Overproduction of eukaryotic SUMO-1- and SUMO-2-conjugated proteins in Escherichia coli. Anal. Biochem. 331, 204–206.
Uchimura, Y., Nakao, M., and Saitoh, H. (2004) Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway. FEBS Lett. 564, 85–90.
Baba, D., Maita, N., Jee, J.G., Uchimura, Y., Saitoh, H., Sugasawa, K., Hanaoka, F., Tochio, H., Hiroaki, H., and Shirakawa, M. (2005) Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature 435, 979–982.
Dadke,S., Cotteret, S., Yip, S.C., Jaffer, Z.M., Haj, F., Ivanov, A., Rauscher, F. 3rd., Shuai, K., Ng, T., Neel, B.G., and Chernoff, J. (2007) Regulation of protein tyrosine phosphatase 1B by sumoylation. Nat. Cell. Biol. 9, 80–85.
Martin, S., Nishimune, A., Mellor, J.R., and Henley, J.M. (2007) SUMOylation regulates kainate-receptor-mediated synaptic transmission. (2007) Nature 447, 321–325.
Uchimura, Y., Ichimura, T., Uwada, J., Tachibana, T., Sugahara, S., Nakao, M., and Saitoh, H. (2006) Involvement of SUMO modification in MBD1- and MCAF1-mediated heterochromatin formation. J. Biol. Chem. 281, 23180–23190.
Lyst, M.J., Nan, X., and Stancheva, I. (2006) Regulation of MBD1-mediated transcriptional repression by SUMO and PIAS proteins. EMBO J. 25, 5317–5328.
Held, D., Yaeger, K., and Novy, R. (2003) New coexpression vectors for expanded compatibilities in E. coli. In Novations 15, 4–6.
Acknowledgments
The authors would like to thank all the members of the Saitoh laboratory. This work was supported by the Naito Foundation and a Grant-in-Aid for Scientific Research from the JSPS to H. S.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Saitoh, H., Uwada, J., Azusa, K. (2009). Strategies for the Expression of SUMO-Modified Target Proteins in Escherichia coli . In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_14
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DOI: https://doi.org/10.1007/978-1-59745-566-4_14
Publisher Name: Humana Press, Totowa, NJ
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