Small Ubiquitin-like Modifier Fusion Research Articles

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein.

African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well.

Extracellular production of Ulp1403-621 in leaky E. coli and its application in antimicrobial peptide production.

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1403-621 approximately fourfold. Moreover, extracellular Ulp1403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1403-621 containing 1mM EDTA and 8mM DTT reached 2.0 × 106 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. KEY POINTS: • First report of extracellular production of Ulp1403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1403-621 approximately fourfold. • The specific activity of extracellular Ulp1403-621 produced in this study reached 2.0 × 106 U/L.

Efficient production of gluten hydrolase Kuma030 in E. coli by hot acid treatment without chromatography

Kumamolisin from Alicyclobacillus sendaiensis strain NTAP-1 is a serine protease with collagenase activity. After molecular engineering, a kumamolisin mutant, named Kuma030, was obtained with high proteolytic activity against gluten, which might cause celiac disease. Kuma030 exhibited its potential application in industrial and medicine, while challenges remained of its large-scale purification and production. In the studies here, we successfully overexpressed the Kuma030 in E. coli BL21 (DE3) by anchoring a SUMO (Small Ubiquitin-like Modifier) fusion protein at its N-terminal end. In addition, a fast protein purification procedure was developed according to the acidophilic and thermophilic properties of Alicyclobacillus sendaiensis. After a simple acid treatment followed by a heat treatment, a total of 9.9 mg functional Kuma030 was quickly obtained form 1 L LB media culture. This purified Kuma030 was confirmed to be functional to cleave the PQ sequences in a designed protein substrate, and the gluten in actual food samples, such as whole wheat bread and beer, in a fast manner. Our studies provided an efficient strategy for the overexpression and purification of functional Kuma030 in E. coli, which might expand its broad practical applications.

Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed &amp; Gwen ColeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optimization of the recombinant production and purification of a self-assembling peptide in Escherichia coli.

BackgroundAmphiphilic peptides are important building blocks to generate nanostructured biomaterials for drug delivery and tissue engineering applications. We have shown that the self-assembling peptide SA2 (Ac-AAVVLLLWEE) can be recombinantly produced in E. coli when fused to the small ubiquitin-like modifier (SUMO) protein. Although this system yielded peptides of high purity with no residual amino acids after cleavage of the SUMO fusion protein, the yield after purification was generally low (~1 mg/L bacterial culture) as compared to other peptides and proteins produced with the same method and under the same conditions.ResultsThe aim of this study is to understand the underlying mechanisms causing the low yield of this recombinant peptide in E. coli and to optimize both production and purification of recombinant SA2 peptides. It was demonstrated that by simply changing the medium to a well-balanced auto-induction medium the yield of recombinant production was augmented (~4 fold). Moreover, it was demonstrated that self-assembly of SUMO-SA2 fusion proteins caused the low peptide yields after purification. By replacing the second IMAC purification step with a selective precipitation step, peptide yields could be increased approx. 3 fold. With these optimizations in place the overall yield of purified SA2 peptide increased with 12-fold.ConclusionPremature self-assembly of the SUMO-SA2 fusion construct interfered with proper purification of the SA2 peptide, resulting in low yields of purified peptide and this could be prevented by changing the mode of purification. These findings are important when setting up purification schemes for other self-assembling peptides with the use of a SUMO fusion construct.

Identification of sumoylated proteins in the silkworm Bombyx mori.

Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori.

Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.

BackgroundScreening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.Methodology/Principal FindingsWe have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.Conclusions/SignificanceThe diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407nm. The enzyme kept its activity between pH 5 and 10 and between 30°C and 60°C, with the highest activity at pH 8.0 and 37°C. The enzyme displayed an apparent Km of 39.08mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.

Purification of recombinant EGFP by fusion with L2 (252–273) from ribosomal protein L2 using magnetic particles

A basic polypeptide L2 (252-273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252-273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252-273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252-273) fusion protein on diatomite was shorter than that of L2 (252-273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252-273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252-273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252-273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252-273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252-273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli.

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

Production of bioactive, SUMO-modified, and native-like TNF-α of the rhesus monkey, Macaca mulatta, in Escherichia coli

Biotechnologically produced tumor necrosis factor alpha (TNF-α) neutralizing agents have proven efficient in patients suffering from disparate autoimmune diseases. The rhesus monkey (Macaca mulatta) could be developed as a model for human autoimmune disease. Consequently, a large amount of M. mulatta TNF-α (mmTNFα) is required to further understand TNF-α-related pathogenesis and evaluate novel human TNF-α (hTNFα) neutralizing agents. We therefore attempted to express mmTNFα by using a small ubiquitin-like modifier (SUMO) fusion system. The synthetic gene, encoding the fusion protein SUMO-mmTNFα, was inserted into a pQE30 plasmid and was transformed into Escherichia coli M15. The fusion protein was expressed as both soluble and insoluble protein in E. coli. Approximately 10-12 mg of SUMO-mmTNFα was obtained from the soluble fraction of 1 L of bacterial culture. Cleavage of the fusion protein with SUMO protease produced native-like mmTNFα. Both native-like and SUMO-modified mmTNFα formed functional trimers and showed excellent cytotoxicity (ED(50), 0.05-0.1 ng/ml) in standard L929 cells. In addition, SUMO-mmTNFα and mmTNFα also exhibited cytotoxicity in human cancer cell types, such as, breast, lung, and liver cancer cells. The hTNFα neutralizing agents, including soluble receptors of hTNFα and antibodies against hTNFα, interacted with the mmTNFα. These results demonstrate that the bioactive mmTNFα produced with the SUMO fusion system is useful for further research, especially for the in vitro preclinical evaluation of biological hTNFα neutralizing agents.

Sumoylation Inhibits Cleavage of Sp1 N-terminal Negative Regulatory Domain and Inhibits Sp1-dependent Transcription

Sp1 is a ubiquitously expressed transcription factor that binds GC-rich cis elements. Many posttranslational modifications have been implicated in the regulation of Sp1 activity. We now provide evidence for a novel mechanism of Sp1 regulation involving the small ubiquitin-like modifier (SUMO-1). Western blot analysis revealed a high molecular mass Sp1 of 125 kDa that is stabilized by a selective SUMO hydrolase inhibitor and destabilized by a specific SUMO-1 hydrolase. The covalent modification of Sp1 by endogenous SUMO-1 and SUMO-1 that has been fused to green fluorescent protein was demonstrated using transient transfection assays. A high probability sumoylation consensus motif, VK(16)IE(18), is located within the N-terminal negative regulatory domain of Sp1. Either arginine substitution for lysine 16 (Sp1(K16R)) or alanine substitution for glutamic acid 18 (Sp1(E18A)), abrogated Sp1 sumoylation. In vitro SUMO-1 covalently bound affinity-purified GST-Sp1, but not GST-Sp1(K16R). In vivo Sp1 was determined to be N-terminally cleaved, while Sp1(K16R) could not be cleaved indicating that sumoylation and cleavage are coupled through the key regulatory lysine 16. This coupling was evident by the demonstration of an inverse relationship between cellular SUMO-modified Sp1 and N-terminally cleaved Sp1. Compared with Sp1, sumoylation-deficient Sp1(E18A) exhibited enhanced cleavage and was a better transcriptional activator, while constitutively SUMO-1-modified Sp1 was deficient in proteolytic processing and repressed Sp1 transcriptional activity. The repressive effect of sumoylation on Sp1 activity is emphasized through the use of a GAL4 based transactivation assay. A model is proposed defining a mechanism by which sumoylation preserves the integrity of a negative regulatory domain thereby allowing for the inhibition of Sp-dependent transcription.