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.     

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH,a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin,is a promising therapeutic candidate for thromboembolic diseases.To develop SFH into a new thrombolytic agent,scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies.Here,we describe a pilot-scale cultivation and purification process for the production of SFH.A high-cell-density fed-batch cultivation for the production of SFH in E.coli was developed in a 40-L bioreactor,which produced about 1.1 g/L of recombinant protein.SFH was purified to homogeneity from the E.coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography,with over 99% purity and 54% recovery.Moreover,the residual endotoxin content was less than 0.5 EU/mL.The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay,respectively.     

Vectors for enhanced gene expression and protein purification in Salmonella

Improved expression vectors have been tested for protein expression studies in Salmonella spp. They are derived from the broad host range expression vector pNSGroE [Seleem, M.N., Vemulapalli, R., Boyle, S.M., Schurig, G.G. and Sriranganathan, N., 2004. Improved expression vector for Brucella species. Biotechniques 37, 740-744] and have several advantages (i) small in size (less than 3 kb); (ii) possess eleven unique restriction site to facilitate directional cloning; (iii) express proteins as His-tagged fusions for easy detection and purification; (iv) carry different promoters for various level of expression and (v) carry an UP element and RNA stem-loop for enhanced gene expression. We have demonstrated the ability of the new vectors to stably express heterologous proteins in Salmonella. We also demonstrated the utility of our vectors by detecting expression and purification of recombinant GFP. Our results suggest that these new vectors should improve gene expression in Salmonella, particularly those aimed at foreign antigen delivery.     

A heme fusion tag for protein affinity purification and quantification

We report a novel affinity‐based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L ‐histidine‐immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme‐tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag‐HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.     

Self-cleaving fusion tags for recombinant protein production

Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.     

Recombinant production of self-assembling SZ-structured peptides using SUMO as a fusion partner

ABSTRACT: BACKGROUND: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P11-family of peptides includes, P11-4 (QQRFEWEFEQQ) and the complementary peptides P11-13 (EQEFEWEFEQE) and P11-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P11-4) or when mixed (P11-13 and P11-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage. RESULTS: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy. CONCLUSIONS: The fusion proteins were produced in high yields and the beta-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.     

GST-talin1融合蛋白的原核表达及纯化

应用PCR方法扩增talin1的cDNA,并将其重组入谷胱甘肽转硫酶融合基因表达载体pGEX-4T-1中,获取人源的GST-talin1融合蛋白,为下阶段深入的研究talin1的结构、功能、及其与之相互作用的蛋白打下基础.经酶切、序列鉴定,选择正确重组子,将其质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,用Glutathione Sepharose 4B柱纯化,western blot鉴定.克隆得到了一个2400bp的talin1的cDNA片断,重组质粒目的DNA测序正确,纯化出分子量约为121.6kD的融合蛋白.用基因工程方法使GST-talin1重组质粒在原核细胞表达并成功纯化出GST-talin1融合蛋白.     

葡萄糖脱氢酶高产工程菌的构建及融合蛋白的一步纯化

PCR扩增葡萄糖脱氢酶基因(glucose dehydrogenase, gdh),连接到测序载体pUC19,转化大肠杆菌JM109,测序(GenBank登录号:EF626962)后,亚克隆到表达载体,转化大肠杆菌M15,筛选得到GDH高产工程菌M15/pQE31-gdh8.工程菌经IPTG诱导表达,超声波破碎,粗酶液比活力高达15 U/mg.镍凝胶柱亲和层析纯化表达蛋白,超滤、冻干后,比活力达360 U/mg.重组质粒pQE31-gdh8在E .coli M15中稳定程度高达99.8%.工程菌M15/pQE31-gdh8诱导表达的重组酶活力高,易纯化,重组质粒稳定程度高,具有良好的工业应用前景.     

SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins

SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides.     

Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

TAT-Cytoglobin融合蛋白的原核表达、纯化及生物活性分析

旨在利用细胞穿膜肽TAT的穿膜作用和细胞珠蛋白(Cytoglobin,Cygb)的抗衰老、抗纤维化的功能,将二者通过基因工程的手段融合在一起,以期获得能够穿透细胞屏障的Cygb。通过两次重叠PCR技术获得了TAT-Cygb DNA,将其插入原核表达载体pET22b质粒中,转化至大肠杆菌BL-21,筛选出可表达TAT-Cygb融合蛋白的大肠杆菌工程菌株。经乳糖诱导表达TAT-Cygb,CM阳离子交换层析(CM Sepharose Fast Flow Protocol)获得纯度高达95%的TAT-Cygb融合蛋白,分子量约23 kDa。生物活性实验显示,TAT-Cygb过氧化物酶比活力达到(422.30±0.36)U/mg。TAT-Cygb预处理的Hacat细胞可免受H2O2氧化应激的损伤(RGR=100%),同时TAT-Cygb可治疗已被H2O2氧化损伤的细胞(RGR=98%),与Cygb处理组相比具有显著差异(RGR=79%)。该研究首次成功利用大肠杆菌表达系统表达了可穿透细胞膜的、有生物活性的TAT-Cygb融合蛋白,为继续开展Cygb在抗衰老、抗纤维化和抗癌领域的研究奠定了基础。     

Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b

The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.     

The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants

Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.