High level expression and purification of recombinant 3ABC non-structural protein of foot-and-mouth disease virus using SUMO fusion system.

The detection of antibody to non-structural protein (NSP) of Foot-and-mouth disease virus (FMDV) is the reliable diagnostic method for differentiating infected from vaccinated animals (DIVA). For this purpose, the detection of antibodies to non-structural 3ABC protein is suitable for identification of virus activity in the animals exposed to FMDV infection. However, large-scale production of recombinant 3ABC protein is challenging due to the formation of inclusion bodies in Escherichia coli and low yield due to protein aggregation during in vitro refolding. In this study, 3ABC gene was fused with SUMO (small ubiquitin-like modifiers) fusion system which significantly enhanced expression of recombinant 3ABC protein in E. coli. The solubility of the recombinant 6xHis-SUMO 3ABC fusion protein was improved by mild detergent treatment and purified through Ni-NTA chromatography under non-denaturing conditions which yielded 9 mg protein obtained from 1-L bacterial fermentation culture. The diagnostic potential of recombinant 3ABC protein was also tested by ELISA that provided reliable diagnostic performance (DSn = 92%, DSp = 94%) upon comparison with commercially available kit. The thermal stability of fusion protein was also tested which presented reliable performance at different temperatures. In conclusion, we presented SUMO fusion for the enhanced expression in E. coli and purification of active recombinant 3ABC protein using non-denaturing conditions without refolding steps. This protein can be used as a suitable diagnostic antigen to detect antibodies following FMDV infection.

Dysregulations of Expression of Genes of the Ubiquitin/SUMO Pathways in an In Vitro Model of Amyotrophic Lateral Sclerosis Combining Oxidative Stress and SOD1 Gene Mutation.

Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.

Cloning and high-level SUMO-mediated fusion expression of a serine protease inhibitor from Hyphantria cunea Drury that exhibits activity against papain.

Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli, resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2-12), temperature (20-50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure.

Expression and purification of an immunogenic SUMO-OmpC fusion protein of Salmonella Typhimurium in Escherichia coli.

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.

Modulation of host cell SUMOylation facilitates efficient development of Plasmodium berghei and Toxoplasma gondii.

SUMOylation is a reversible post translational modification of proteins that regulates protein stabilization, nucleocytoplasmic transport, and protein-protein interactions. Several viruses and bacteria modulate host SUMOylation machinery for efficient infection. Plasmodium sporozoites are infective forms of malaria parasite that invade mammalian hepatocytes and transforms into exoerythrocytic forms (EEFs). Here, we show that during EEF development, the distribution of SUMOylated proteins in host cell nuclei was significantly reduced and expression of the SUMOylation enzymes was downregulated. Plasmodium EEFs destabilized the host cytoplasmic protein SMAD4 by inhibiting its SUMOylation. SUMO1 overexpression was detrimental to EEF growth, and insufficiency of the only conjugating enzyme Ubc9/E2 promoted EEF growth. The expression of genes involved in suppression of host cell defense pathways during infection was reversed during SUMO1 overexpression, as revealed by transcriptomic analysis. The inhibition of host cell SUMOylation was also observed during Toxoplasma infection. We provide a hitherto unknown mechanism of regulating host gene expression by Apicomplexan parasites through altering host SUMOylation.

Activating transcription factor 3 SUMOylation is involved in angiotensin II-induced endothelial cell inflammation and dysfunction.

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli.

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A.

Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.

The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology.

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis.

Lacticin Q is a 53-amino acid Class II bacteriocin produced by Lactococcus lactis QU 5. It shows antibacterial activity comparable to that of nisin A in terms of both spectrum and intensity. Moreover, it remains stable at alkaline pH values, while nisin A was inactivated. It may possibly be employed as an alternative to or in combination with nisin A. The objective of this study was to express lacticin Q extracellularly with Small ubiquitin-related modifier (SUMO) fusion technology in Bacillus subtilis. Secretory SUMO-lacticin Q fusion protein was efficiently produced in B. subtilis WB600 transformed with the recombinant expression plasmid and accounted for 19% of the culture supernatant proteins. Fusion SUMO-lacticin Q was purified by nickel nitrilotriacetate (Ni-NTA) affinity chromatography and digested with SUMO protease to release lacticin Q. Lacticin Q was further purified by Ni-NTA chromatography to yield about 2.5 mg of lacticin Q with more than 93% purity from 1L of supernatant of fermentation culture. An activity assay indicated that the recombinant bacteriocin exhibited excellent antimicrobial activity against indicator strains. The results obtained suggest that the secretory lacticin Q was efficiently expressed using SUMO fusion technology in B. subtilis. The expression and purification system could promote the application of lacticin Q in food and medicine.

A SUMO-Groucho Q domain fusion protein: characterization and in vivo Ulp1-mediated cleavage.

We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.

Production of recombinant peptides as fusions with SUMO.

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.

SUMO mediating fusion expression of antimicrobial peptide CM4 from two joined genes in Escherichia coli.

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. To improve the expression level of CM4 in Escherichia coli, two tandem repeats of CM4 genes were cloned into the vector pSUMO to construct an expression vector pSUMO-2CM4. The fusion protein SUMO-2CM4, purified by Ni(2+)-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. After the cleaved sample was re-applied to a Ni-IDA column, finally, about 48 mg recombinant CM4 was obtained from 1 L bacterial culture with no less than 96% purity, which was the highest yield of CM4 reported so far.

Enrichment of fermentation media and optimization of expression conditions for the production of EAK(16) peptide as fusions with SUMO.

EAK(16) (AEAEAKAKAEAKAEAK) belongs to a novel class of self-assembling peptides, which is being investigated in research and industry. SUMO belongs to the ubiquitin class of proteins and is a promising fusion partner currently in use. In this study, EAK(16) peptide fusions with hexa-histidine tagged SUMO have been constructed using Escherichia coli based pET expression vector. Intracellular expression of the SUMO-EAK(16) fusion using LB media has been optimized. Low-cost complex media (fungal autolysates, wheat and gluten hydrolysates) produced via a novel wheat-based biorefinery have been used as alternative fermentation media to LB. Shake flask cultures using either enriched LB or complex wheat-derived media containing 2 g/L of glucose resulted in intracellular SUMO-EAK(16) fusion protein production of approximately 250 mg/L fermentation volume which corresponded to 30-35% of the total bacterial protein expressed being the fusion protein. Fusion protein productivities up to five times higher were achieved when using a bioreactor.

SUMO1P3 is associated clinical progression and facilitates cell migration and invasion through regulating miR-136 in non-small cell lung cancer.

Long non-coding RNA small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) is located on chromosome 1q23.2, and has been suggested to serve as oncogenic lncRNA in many kinds of human malignancy. The role of SUMO1P3 in non-small cell lung cancer (NSCLC) was still unknown. In our study, we analyzed The Cancer Genome Atlas (TCGA) database, and observed SUMO1P3 expression was increased in both lung squamous cell carcinoma and lung adenocarcinoma. Then, we confirmed that SUMO1P3 expression was significantly increased in NSCLC cancer tissues and cell lines. Meanwhile, the expression levels of SUMO1P3 expression in metastatic lymph node specimens were strikingly elevated in comparison to primary NSCLC tissue specimens. Then, we found high SUMO1P3 expression was correlated with late clinical stage, lymph node metastasis, distant metastasis and poor differentiated degree. In the survival analysis of TCGA, we observed that SUMO1P3 expression had no association with overall survival and disease free survival in NSCLC patients. There was a statistically negative correlation between SUMO1P3 expression and miR-136 expression in NSCLC tissues. Moreover, miR-136 directly bound to SUMO1P3, and SUMO1P3 negatively regulated miR-136 expression in NSCLC cells. Furthermore, SUMO1P3 promoted NSCLC cell migration and invasion via regulating miR-136. In conclusion, SUMO1P3 functions as metastasis-associated lncRNA in NSCLC.

SUMO-1 overexpression increases RbAp46 protein stability and suppresses cell growth.

The retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46) is a nuclear protein of 46 kDa and contains four repeats that end with Trp-Asp (WD) residues. In this study, we reveal that the RbAp46 protein level upon SUMO-1 expression was increased. The increasing level of RbAp46 protein by SUMO-1 was not regulated at the transcriptional level. SUMO-1 does not affect the degradation of RbAp46. Co-localization of RbAp46 and SUMO-1 in the nuclei of stable NIH/3T3 cells harboring the inducible Ha-ras(Val12) oncogene (pSVlacOras) designated as 7-4, and protein-protein interaction between RbAp46 and SUMO-1 was also detected by co-immunoprecipitation in these cells. However, SUMO-l-related sumoylation was not involved in the modification of RbAp46. It is possibly that SUMO-1 acts through formation of complex with RbAp46 to stabilize RbAp46 protein. Overexpression of RbAp46 protein suppressed the NIH/3T3 cell growth induced by Ha-Ras(V12). SUMO-1 further enhances the suppression of cell growth through stabilization of RbAp46 protein. This is the first report to demonstrate that SUMO-1 can suppress Ras-related cell proliferation through stabilization of RbAp46 protein.

Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System.

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.

Production of sumoylated proteins using a baculovirus expression system.

Spodoptera frugiperda Sf9 cells were found to possess an active endogenous sumoylation system. However, the endogenous sumoylation machinery did not efficiently modify exogenous proteins expressed by infection with recombinant baculoviruses. To overcome this limitation, mammalian sumoylation components were introduced by co-infection with recombinant baculoviruses expressing individual protein components of the sumoylation pathway. Expression of mammalian Ubc9 plus SUMO (either SUMO1 or SUMO3) was necessary and sufficient for active sumoylation of co-infected test proteins. This system provides a simple and convenient means to produce sumoylated mammalian proteins in a eukaryotic environment. Large-scale cultures should provide quantities of sumoylated proteins sufficient for potential purification.

High Resolution X-ray Diffraction Dataset for Bacillus licheniformis Gamma Glutamyl Transpeptidase-acivicin complex: SUMO-Tag Renders High Expression and Solubility.

Gamma glutamyl transpeptidase, (GGT) is a ubiquitous protein which plays a central role in glutathione metabolism and has myriad clinical implications. It has been shown to be a virulence factor for pathogenic bacteria, inhibition of which results in reduced colonization potential. However, existing inhibitors are effective but toxic and therefore search is on for novel inhibitors, which makes it imperative to understand the interactions of various inhibitors with the protein in substantial detail. High resolution structures of protein bound to different inhibitors can serve this purpose. Gamma glutamyl transpeptidase from Bacillus licheniformis is one of the model systems that have been used to understand the structure-function correlation of the protein. The structures of the native protein (PDB code 4OTT), of its complex with glutamate (PDB code 4OTU) and that of its precursor mimic (PDB code 4Y23) are available, although at moderate/low resolution. In the present study, we are reporting the preliminary analysis of, high resolution X-ray diffraction data collected for the co-crystals of B. licheniformis, Gamma glutamyl transpeptidase, with its inhibitor, Acivicin. Crystals belong to the orthorhombic space group P212121 and diffract X-ray to 1.45 Å resolution. This is the highest resolution data reported for all GGT structures available till now. The use of SUMO fused expression system enhanced yield of the target protein in the soluble fraction, facilitating recovery of protein with high purity. The preliminary analysis of this data set shows clear density for the inhibitor, acivicin, in the protein active site.

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

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 fluorescent 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 KM values, but SUMO protease had a 25-fold higher kcat 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.