HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: ps.051812706v1 15/1/182    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marblestone, J. G. Articles by Butt, T. R. Search for Related Content PubMed PubMed Citation Articles by Marblestone, J. G. Articles by Butt, T. R. Social Bookmarking                   What's this?

Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO

¹ Progenra Inc., Malvern, Pennsylvania 19355, USA
² LifeSensors Inc., Malvern, Pennsylvania 19355, USA

(RECEIVED August 26, 2005; FINAL REVISION October 12, 2005; ACCEPTED October 12, 2005)

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green florescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMOtags.These constructswere expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP,MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similarK_M values, but SUMO protease had a 25-fold higher k_cat than AcTEV protease, indicating amore catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.

Keywords: fusion protein; protease; structural genomics; SUMO; ubiquitin-like protein

Abbreviations: DUB, deubiquitinating enzyme or ubiquitin specific protease/hydrolase • eGFP, enhanced green fluorescent protein • IPTG, isopropropyl--D-thiogalactopyranoside • MBP, E. coli maltose-binding protein • Ni-NTA, nickel-nitrilotriacetic acid • PCR, polymerase chain reaction • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • Ub, ubiquitin • Ubl(s), ubiquitin-like protein(s) • ULP, catalytic domain of Ulp1

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051812706.

Reprint requests to: Tauseef R. Butt, LifeSensors Inc., 271 Great Valley Parkway, Malvern, PA 19355, USA; e-mail: butt{at}lifesensors.com; fax: (610) 644-8616.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. E. Duncan, E. Sarkadi-Nagy, K. Jaworski, M. Ahmadian, and H. S. Sul Identification and Functional Characterization of Adipose-specific Phospholipase A2 (AdPLA) J. Biol. Chem., September 12, 2008; 283(37): 25428 - 25436. [Abstract] [Full Text] [PDF]

				R. J. Peroutka, N. Elshourbagy, T. Piech, and T. R. Butt Enhanced protein expression in mammalian cells using engineered SUMO fusions: Secreted phospholipase A2 Protein Sci., September 1, 2008; 17(9): 1586 - 1595. [Abstract] [Full Text] [PDF]

				C.-D. Lee, H.-C. Sun, S.-M. Hu, C.-F. Chiu, A. Homhuan, S.-M. Liang, C.-H. Leng, and T.-F. Wang An improved SUMO fusion protein system for effective production of native proteins Protein Sci., July 1, 2008; 17(7): 1241 - 1248. [Abstract] [Full Text] [PDF]

				B. Nicholson, C. A. Leach, S. J. Goldenberg, D. M. Francis, M. P. Kodrasov, X. Tian, J. Shanks, D. E. Sterner, A. Bernal, M. R. Mattern, et al. Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities Protein Sci., June 1, 2008; 17(6): 1035 - 1043. [Abstract] [Full Text] [PDF]