Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli

Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.     

Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method

Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.     

Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli

A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.     

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.     

Optimized procedures for producing biologically active chemokines

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6×His-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni–NTA–agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6×His-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni–NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.     

High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile β-trefoil lectin domain from the mushroom Laetiporus sulphureus

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47?? resolution and also that of the [LSL(150):(lactose)β, γ)] binary complex at 1.67?? resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.     

Screening of fusion partners for high yield expression and purification of bioactive viscotoxins

Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2mg of visctoxin A3 from 1l of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.     

New fusion protein systems designed to give soluble expression in Escherichia coli.

Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.     

His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors

We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.     

Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli

High expression of recombinant proteins in Escherichia coli (E. coli) often leads to protein aggregation. One popular approach to address this problem is the use of fusion tags (or partners) that improve the solubility of the proteins in question. However, such fusion tags are not effective for all proteins. In this study, we demonstrate that the hyper-acidic protein fusion partners can largely enhance the soluble expression of target proteins recalcitrant to the efforts by using routine solubilising tags. This new type of fusion partners examined includes three extremely acidic E. coli polypeptides, i.e. yjgD, the N-terminal domain of rpoD (sigma 70 factor of RNA polymerase) and our preliminarily evaluated msyB. The target proteins used are highly aggregation-prone, including EK (the bovine enterokinase), TEV (the tobacco etch virus protease) and rbcL (the large subunit of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase). On removal in vitro and in vivo of the fusion tags by using yeast SUMO/Ulp1 reaction and TEV auto-cleavage, the resultant findings indicate the hyper-acidic fusion partners can function as intramolecular chaperones assisting in the correct folding of the target proteins.     

A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.     

A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy

The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.     

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.     

A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions

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 Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. 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 His(6)-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.     

高活性重组hG-CSF的制备

我们构建了新的硫氧还蛋白（Ｔｈｉｏｒｅｄｏｘｉｎ）融合表达载体ｐＥＴＴｒｘＬ和ｐＥＴＴｒｘ－ＨｉｓＬ,它们可使功能蛋白在大肠杆菌胞质中以可溶性形式高效表达。利用此表达系统成功地获得的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白的高效可溶性表达,表达水平达总细胞可溶蛋白的４１％以上。所表达的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白可通过Ｃｕ２＋－ＩＤＡＳｅｐｈａｒｏｓｅＦＦ固相金属螯合层析柱,方便地从细胞破碎可溶上清中直接纯化。所获得的融合蛋白具有ｈＧ－ＣＳＦ特异的生物活性,其比活性达到０．５－１．３３×１０７ｕ／ｍｇ融合蛋白。这样表达的ｈＧ－ＣＳＦ融合蛋白能被ＩｇＡ蛋白酶特异地切割,将ｈＧ－ＣＳＦ从融合蛋白上切下获得与天然蛋白一级结构完全一致的重组ｈＧ－ＣＳＦ 。     

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.     

Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems

In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.