Enhanced Expression and Purification of Membrane Proteins by SUMO Fusion in Escherichia coli (original) (raw)
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Expression and purification of SARS coronavirus proteins using SUMO-fusions
Protein Expression and Purification, 2005
Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is diYcult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitinrelated modiWer), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6£ His-tagged SUMO-fusions facilitate rapid puriWcation of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates puri-Wcation, and can be eYciently cleaved by a SUMO-speciWc protease to generate native protein with a desired N-terminus. We have puriWed various SARS-CoV proteins under either native or denaturing conditions. These puriWed proteins have been used to generate highly speciWc polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and puriWcation of the viral proteins for structural and functional studies as well as for therapeutic uses. 2005 Published by Elsevier Inc.
Enhanced Expression of Rabies Virus Surface G-Protein in Escherichia coli using SUMO Fusion
The Protein Journal, 2012
Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased *1.5 fold higher expression and *3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research. Keywords Enhanced expression Á Rabies glycoprotein Á Recombinant protein Á Solubility Á SUMO fusion Abbreviations FLAP 5-lipoxygenase-activating protein rgp Rabies glycoprotein gene RGP Rabies glycoprotein SARS-CoV Severe acute respiratory syndrome coronavirus SUMO Small ubiquitin-like modifier S-RGP SUMO fusion with rabies glycoprotein UBL Ubiquitin-like modifiers UDPs Ubiquitin-domain proteins Electronic supplementary material The online version of this article (
Methods in molecular biology (Clifton, N.J.), 2011
The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility ( http://www.lifesensors.com ). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes.
SUMO fusion technology for difficult-to-express proteins
Protein Expression and Purification, 2005
The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the Weld of protein production and puriWcation. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good puri-Wcation methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, puriWcation, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems. 2005 Published by Elsevier Inc.
Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions
Protein expression and purification, 2008
Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in E. coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous de-SUMOylases. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15 and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His6 tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.
SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins
Journal of Structural and Functional Genomics, 2000
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 ϩ 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.
Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli
2024
The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.
MAKARA of Science Series, 2011
Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, we reported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E. coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chain reaction (RT-PCR) technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the target gene into pQE-80L. After inserting the recombinant plasmid (pQE80-N) into E. coli, the recombinant protein (6 x His tag-N protein fusion) was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-Dgalactopyranoside (IPTG) for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffer containing 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction of E. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mM imidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.
An improved SUMO fusion protein system for effective production of native proteins
Protein Science, 2008
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 59-end and XhoI at the 39-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.
Microbial Cell Factories, 2011
Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm) or degraded (cytoplasm) whereas at 18°C, expression was improved especially in the periplasm of C41(DE3) cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3) cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This hypothesis is supported by the fact that use of minimal media could enhance protein production compared to nutrient-rich LB media. We have found optimal conditions for heterologous expression of U24 from Human Herpesvirus type-6 in E.coli and have demonstrated that milligram quantities of pure protein can be obtained. Strained metabolic conditions such as low temperature, minimal media and an oxidizing environment appeared essential for high-level, full-length protein production and this information may be useful for expressing other membrane proteins of interest.