Small ubiquitin-like modifier (SUMO) fusion tag
Small ubiquitin-like modifier (SUMO) fusion tag, the 98-residue yeast Smt3 protein, placed N-terminal to a target protein to increase its yield and solubility during recombinant expression. A SUMO protease cleaves at the C terminus of the tag and leaves no additional amino acids on the target.
Origin: Saccharomyces cerevisiae SUMO (Smt3)
Characteristics
Encodes the 98-amino-acid yeast SUMO (Smt3), beginning MSDSEVNQ and ending AHREQIGG, the Gly-Gly motif after which SUMO protease cleaves. SUMO is about 11.2 kDa. SUMO protease recognizes the tertiary structure of the tag, not a linear peptide sequence, and cleaves precisely at the C terminus of the tag, so the target keeps its authentic N terminus. The yeast Ulp1 catalytic domain cleaved SUMO-GFP with any residue except proline after the Gly-Gly, and was active at pH 6 to 10 and 4 to 37 °C. SUMO protease kcat is reported to be about 25-fold higher than TEV protease.
Applications: Expression of cytokines (IL-1 beta, IL-8, IFN-gamma, TNF-alpha) as active proteins, and of 15 chemokines with authentic N termini. His6-SUMO gave cyanovirin-N at over 30% of total soluble protein in E. coli in a 30 L bioreactor, and made MMP13, insoluble as a His6 fusion, mostly soluble in E. coli. Current SUMO tags are used in E. coli, Pichia pastoris, and mammalian HEK and CHO cells.
Limitations: Tag removal needs a SUMO protease, an added reagent and purification step, and that protease removes only SUMO tags. Proline as the first residue of the target blocks cleavage. SUMO C-terminal to GFP did not enhance expression. SUMO did not improve solubility of 15 chemokines.
Sequence
References
- Malakhov et al. (2004). SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics - Malakhov 2004 SUMO Fusions
- Bell et al. (2013). To fuse or not to fuse: what is your purpose?. Protein Sci - Bell 2013 Fusion Purpose