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

Abstract

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 SUMO tags. These constructs were 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 similar K M values, but SUMO protease had a 25-fold higher k cat than AcTEV protease, indicating a more 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.

PML nuclear bodies: Assembly and oxidative stress-sensitive sumoylation

“…26,90,91 Various exogenous factors, such as heat or oxidative stress cause protein misfolding or aggregation, but also hypersumoylation. [94][95][96] Thus, ROS/NB-facilitated hyper-sumoylation could provide a rapid first line protection against protein aggregation. Proteins irreversibly damaged could then be channelled into the RNF4/Ubiquitin pathway, and eventually degraded by the nuclear proteasome which accumulates around PML NBs 28,97 The mechanistic basis of how misfolded proteins are recognized by PML needs further exploration.…”

Soluble expression and purification of heparinase I in Escherichia coli using a hexahistidine-tagged small ubiquitin-like modifier as a fusion partner

“…Soluble partners are able to enhance the solubility of recombinant protein, such as glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), etc [12,13]. Among the expression partners, SUMO, a small ubiquitin-modifying protein which can dramatically improve the soluble expression, has attracted a lot of attention recently [14,15]. A comparison between SUMO fusion technologies and traditional gene fusion systems implies that SUMO is superior to traditional fusion tags in enhancing protein expression and solubility [14].…”

Structure of Est3 reveals a bimodal surface with differential roles in telomere replication

“…aggregates. To overcome this, extensive optimization of the protein construct and sample conditions as well as multiple solubility enhancing tags were investigated (13,14), with the best results obtained with the fusion of a His 10 -SUMO tag to the N terminus of the Est3 protein (15). Still, the 15 N-HSQC spectrum and heteronuclear NOE (HetNOE) NMR measurement of Est3 suggested the presence of a disordered region within the fulllength protein (Fig.…”