SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems
In eukaryotic cells, the reversible attachment of small ubiquitin-like modifi er (SUMO) protein is a post-translational modifi cation that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner enhances functional protein production in prokaryotic and eukaryotic expression systems, based upon significantly improved protein stability and solubility. Following the expression and purification of the fusion protein, the SUMO-tag can be cleaved by specific (SUMO) proteases via their endopeptidase activity in vitro to generate the desired N-terminus of the released protein partner. In addition to its physiological relevance in eukaryotes, SUMO can, thus, be used as a powerful biotechnological tool for protein expression in prokaryotic and eukaryotic cell systems.
In this chapter, we will describe the construction of a fusion protein with the SUMO-tag, its expression in Escherichia coli, and its purification followed by the removal of the SUMO-tag by a SUMO-specific protease in vitro.
Key wordsSUMO fusion Ulp1 protein expression protein stability solubility
- 2.Meinecke, I., Cinski, A., Baier, A., Peters, M. A., Dankbar, B., Wille, A., Drynda, A., Mendoza, H., Gay, R. E., Hay, R. T., Ink, B., Gay, S., and Pap, T. (2007) Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts. Proc. Natl. Acad. Sci. USA 104, 5073–5078.PubMedCrossRefGoogle Scholar
- 10.Zuo, X., Mattern, M. R., Tan, R., Li, S., Hall, J., Sterner, D. E., Shoo, J., Tran, H., Lim, P., Sarafianos, S. G., Kazi, L., Navas-Martin, S., Weiss, S. R., and Butt, T. R. (2005) Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J. Struct. Funct. Genomics 6, 103–111.PubMedCrossRefGoogle Scholar
- 11.Zuo, X., Mattern, M. R., Tan, R., Li, S., Hall, J., Sterner, D. E., Shoo, J., Tran, H., Lim, P., Sarafianos, S. G., Kazi, L., Navas-Martin, S., Weiss, S. R., and Butt, T. R. (2005) Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr. Purif. 42, 100–110.PubMedCrossRefGoogle Scholar
- 14.Hughes, R. S., Dowd, P. F., Hector, R. E., Riedmuller, S. B., Bartolett, S., Mertens, J. A., Qureshi, N., Liu, S., Bischoff, K. M., Li, X., Jackson Jr., J. S., Sterner, D., Panavas, T., Rich, J. O., Farrelly, P. J., Butt, T., and Cotta, M. A. (2007) Cost-Effective High-Throughput Fully Automated Construction of a Multiplex Library of Mutagenized Open Reading Frames for an Insecticidal Peptide Using a Plasmid-Based Functional Proteomic Robotic Workcell with Improved Vacuum System. J. Assoc. Lab. Autom. 12, 202–212.CrossRefGoogle Scholar
- 16.Liu, L., Spurrier, J., Butt, T. R., Strickler, J.E. (2008) Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions. Protein Expr. Purif. Epub ahead of print 5 Aug 2008.Google Scholar