Human SUMO Fusion Systems Enhance Protein Expression And Solubility

Escherichia coli is the preferred host for rapid and low-cost production of recombinant proteins and peptides for biochemical analysis, therapeutics or structural studies. Even though recently great progresses have been made in E. coli protein production, expression of heterogeneous proteins in such a host has its attendant problems, such as protein misfolding and insolubility. Several methods, including promoter alterations, chaperone co-expression and protein fusions, have been used to circumvent these problems. Unfortunately, these methods are mostly for specific proteins and cannot be widely used.Fusion systems can not only facilitate protein expression and folding, but also prevent protein degradation and simplify purification and detection. As such, protein fusions have generally been used to improve the expression and solubility of recombinant proteins. Examples of fusion partners include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (Trx), N-utilization substance A (NusA), ubiquitin (Ub), and yeast SUMO (small ubiquitin-related modifier). These tags have been successfully used in the heterologous expression of correctly folded and soluble passenger proteins in E. coli. Recently, several difficult-to-express proteins, including severe acute respiration syndrome coronavirus (SARS-CoV) 3CL protease, enhanced green fluorescent protein (eGFP), human matrix metalloprotease-13 (MMP13), myostatin (growth differentiating factor-8, GDF8) and membrane proteins, have been successfully expressed by a yeast SUMO fusion system. In addition, the yeast SUMO fusion system has a distinguishing feature: SUMO protease could cleave the fusion proteins with remarkable fidelity and efficiency, and could generate passenger proteins with natural N-terminal amino acid. Yeast has a single SUMO gene (SMT3), while in humans there are three SUMO genes (SUMO1, SUMO2, SUMO3) that are highly homologous. SUMO1 shares 50% sequence identity with SUMO2 and SUMO3, while SUMO-2 shares 87% sequence identity with SUMO-3. SUMO1 shares greater sequence identity with yeast SUMO (47%) than SUMO2 and SUMO3. Although yeast SUMO has achieved great success as a fusion tag, the effects of human SUMOs on protein expression have not been investigated.In the present paper, we tested a novel human SUMO (SUMO1 & SUMO2) fusion expression system for high-level expression and production of soluble authentic recombinant proteins. Two candidate proteins, human matrix metalloprotease-13 (MMP13, amino acid 104-274) and enhanced green fluorescent protein (eGFP) were expressed as fusion proteins with His6, ubiquitin (Ub), SUMO1 and SUMO2. These constructs were expressed in E. coli and evaluated for expression and solubility. It was found that fusion of SUMO1 and SUMO2 improved the solubility of MMP13 and enhanced expression and solubility of eGFP. Remarkably, SUMO2, which shares less sequence identity with yeast SUMO than SUMO1, had a greater effect. This may indicate that SUMO2 may have a better effect than yeast SUMO on protein expression and solubility.Since both ubiquitin and human SUMO proteins can improve protein expression and solubility, we also tested whether SUMO2 coupled with ubiquitin had a better effect when used as a tag, and whether the sequential order of SUMO2 and ubiquitin had any impact on protein expression and solubility. The result indicated that the solubility of SUMO2-ubiquitin-eGFP and ubiquitin-SUMO2-eGFP was better than ubiquitin-eGFP and was similar to that of SUMO2-eGFP. There was no additive effect noted with no effect on expression and solubility based on the sequential order of SUMO2 and ubiquitin in tandem constructs. Also, we want to test whether our expression system can facilitate protein purification. Using readily available techniques of protein purification (first purified by IMAC, then enzymatic cleavage and finally purified by IMAC again), the recombinant eGFP protein was easily produced with high homogeneity and activity. In summary, we describe two novel fusion tags (human SUMO1 and SUMO2) that improved the expression and solubility of difficult-to-express passenger proteins. In all constructs, human SUMO2, with less sequence identity to yeast SUMO, had a better effect than human SUMO1. This report indicates that human SUMO protein fusion systems may present a viable alternative for the soluble expression of proteins that have been impossible using traditional gene fusion technologies.