SpringerProtocols: Abstract: SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems

20. SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems

By: Tadas Panavas³, Carsten Sanders³, Tauseef R. Butt³

Abstract

Full Text

Download PDF (434K)

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.

Affiliation(s): (3) Life Sensors Inc., Malvern, PA, USA

Book Title: SUMO Protocols

Series: Methods in Molecular Biology | Volume: 497 | Pub. Date: Oct-31-2008 | Page Range: 303-317 | DOI: 10.1007/978-1-59745-566-4_20

Subject: Biochemistry

Key Words: SUMO fusion - Ulp1 - protein expression - protein stability - solubility

References

Download References

1.	Seeler, J. S., Bischof, O., Nacerddine, K., and Dejean, A. (2007) SUMO, the three Rs and cancer. Curr. Top. Microbiol. Immunol. 313, 49–71.

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.

3.	Rajan, S., Plant, L. D., Rabin, M. L., Butler, M. H., and Goldstein, S. A. (2005) Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell 121, 37–47.

4.	Martin, S., Nishimune, A., Mellor, J. R., and Henley, J. M. (2007) SUMOylation regulates kainate-receptor-mediated synap-tic transmission. Nature 447, 321–325.

5.	Mabb, A. M., Wuerzberger-Davis, S. M., and Miyamoto, S. (2006) PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress. Nat. Cell Biol. 8, 986–993.

6.	Li, S. J. and Hochstrasser, M. (1999) A new protease required for cell-cycle progression in yeast. Nature 398, 246–251.

7.	Malakhov, M. P., Mattern, M. R., Mala-khova, O. A., Drinker, M., Weeks, S. D., and Butt, T. R. (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J. Struct. Funct. Genomics 5, 75–86.

8.	Marblestone, J. G., Edavettal, S. C., Lim, Y., Lim, P., Zuo, X., and Butt, T. R. (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 15, 182–189.

9.	Butt, T. R., Edavettal, S. C., Hall, J. P., and Mattern, M. R. (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr. Purif. 43, 1–9.

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.

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.

12.	Guzzo, C. M. and Yang, D. C. (2007) Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli. Protein Expr. Purif. 54, 166–175.

13.	Dominy, J. E., Jr., Simmons, C. R., Hirsch-berger, L. L., Hwang, J., Coloso, R. M., and Stipanuk, M. H. (2007) Discovery and characterization of a second mammalian thiol dioxygenase, cysteamine dioxygenase. J. Biol. Chem. 282, 25189–25198.

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.

15.	Peroutka, R., Elshourbagy, J., N., Piech, T., and Butt, T.R. (2008) Enhanced protein expression in mammalian cells using engineered SUMO fusion: Secreted Phospholi-pase A₂. Protein Sci. 17, 1586–1595.

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.

17.	Mossessova, E., and Lima, C. D. (2000) Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876.

Comments (Loading...)

Post comment/View all comments