Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli

Solubility of proteins on overexpression in Escherichia coli is a manifestation of the net effect of several sequence-dependent and sequence-independent factors. This study aims to delineate the relationship between the primary structure and solubility on overexpression. The amino acid sequences of proteins reported to be soluble or to form inclusion bodies on overexpression in E. coli under normal growth conditions were analyzed. The results show a positive correlation between thermostability and solubility of proteins, and an inverse correlation between the in vivo half-life of proteins and solubility. The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein were also found to influence its solubility on overexpression. The amino acids that were seen to be present in a comparatively higher frequency in inclusion body-forming proteins have a higher sheet propensity, whereas those that are seen more in soluble proteins have a higher helix propensity; this is indicative of a possible correlation between sheet propensity and inclusion body formation. Thus, the present analysis shows that thermostability, in vivo half-life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli. The precise mechanism by which these properties affect the solubility status of the overexpressed protein remains to be understood.     

Correlation between the structural stability and aggregation propensity of proteins

Protein aggregation, being an outcome of improper protein folding, is largely dependent on the folding kinetics of a protein. Previous studies have reported a positive correlation between the stability of the secondary structural elements of a protein and their rate of folding/unfolding. In this in silico study, the secondary and tertiary structures of proteins a) that form inclusion bodies on overexpression in Escherichia coli, b) that form amyloid fibrils and c) that are soluble on overexpression in E. coli are analyzed for certain features that are known to be associated with structural stability. The study revealed that the soluble proteins seem to have a higher rate of folding (based on contact order) and a lower percentage of exposed hydrophobic residues as compared to the inclusion body forming or amyloidogenic proteins. The soluble proteins also seem to have a more favored helix and strand composition (based on the known secondary structural propensities of amino acids). The secondary structure analyses also reveal that the evolutionary pressure is directed against protein aggregation. This understanding of the positive correlation between structural stability and solubility, along with the other parameters known to influence aggregation, could be exploited in the design of mutations aimed at reducing the aggregation propensity of the proteins.     

Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli.

Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.     

In vivo increase of solubility of overexpressed Hha protein by tandem expression with interacting protein H-NS

Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli.     

A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy.     

Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins

Proteins and peptides expressed in the prokaryotic system often form inclusion bodies. Solubilization and refolding procedures can be used for their recovery, but this process remains difficult. One strategy for improving the solubility of a protein of interest is to fuse it to a highly soluble protein. To select a suitable fusion partner capable of solubilizing the aggregation-prone (inclusion body-forming) proteins and peptides, Escherichia coli thermostable proteins were identified and tested. Among them, trigger factor (TF) protein was selected because of its high expression and stability. Using an expression system based on fusion to TF, selected proteins and peptides that otherwise form inclusion bodies were expressed in soluble state and were purified like other soluble proteins. This system provides a convenient method for production of aggregation-prone proteins and peptides.     

Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.     

High-yield production of secreted active proteins by the Pseudomonas aeruginosa type III secretion system

The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.     

A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment

Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product that needs to be refolded. By screening the solubility pattern for a set of 71 target proteins in different host-strains and varying parameters such as location of purification tag, promoter and induction temperature we propose a protocol with a success rate of 77% of clones returning a soluble protein. This protocol is particularly suitable for high-throughput screening with the goal to obtain soluble protein product for e.g. structure determination.     

DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli

Overexpression of CorA, the major magnesium transporter from bacterial inner membrane, in Escherichia coli resulted in the synthesis of 60mg of protein per liter of culture, most of which however was in the form of inclusion bodies. The levels of inclusion body formation were reduced by lowering the cell culture temperature. To dissect CorA inclusion body formation and the folding process involved, we co-expressed the protein with various chaperones and other folding modulators. Expression of DnaK/DnaJ (Hsp70) prevented inclusion bodies from forming and resulted in the integration of more CorA into the membrane. GroEL/GroES (Hsp60/Hsp10) were less effective at reducing CorA inclusion body formation. Co-expression with either Ffh/4.5S-RNA, the signal recognition particle, or SecA, the ATPase that drives protein insertion into the membrane, had little effect on CorA folding. These results indicate: (1) that CorA inclusion bodies form immediately after synthesis at 37 degrees C, (2) that CorA solubility in the cytosol can be increased by co-expressing a chaperone system, (3) membrane targeting is probably not a rate-limiting factor, and (4) that membrane insertion becomes a limitation only when large amounts of soluble CorA are present in the cytosol. These co-expression systems can be used for producing other membrane proteins in large quantities.     

Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli

Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a single-chain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might be applicable for high-yield soluble expression of proteins with multiple disulfide bonds.     

Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners

Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.     

A novel high-cell-density protein expression system based on Ralstonia eutropha

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.     

Protein aggregation as bacterial inclusion bodies is reversible

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.     

Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli

The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.     

Enhancement of the solubility of proteins overexpressed in Escherichia coli by heat shock

Protein misfolding resulting in the formation of inclusion bodies is one of the major problems during protein overexpression in Escherichia coil. In this paper, we introduce a new method, which is simply to heat shock a cell culture prior to protein induction, allowing effective enhancement of the solubility and thereby the yield of overexpressed proteins in E. coli. Using this method, we show that the solubility of the E. coli protein KsgA-AN is significantly increased when overexpressed from a T7 promoter. In addition, we also show that the solubility of several Caenorhabditis elegans proteins are also enhanced after heat-shock treatment when expressed in E. coli. Taken together, these results suggest that the "heat-shock protocol" is a generalizable and useful method for increasing the solubility of many proteins overexpressed in E. coli.     

Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase

Recombinant mammalian proteins expressed in E. coli can be difficult to purify in high yield in a soluble and functional form. Various techniques have been described to prevent proteolysis of expressed proteins and/or their sequestering as insoluble aggregates within inclusion bodies. We report conditions for expressing recombinant proteins from E. coli that significantly enhanced the yield of soluble and functional protein. We demonstrate high-yield recovery of a native, high-molecular-weight RNA binding protein without the aid of fusion protein sequence. The principle factor that increased protein yield was the induction of protein expression in a late log phase culture, although reduced temperature during the induction and a low IPTG concentration also contributed to a higher yield.     

Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli

Although large amounts of wild-type human Cu,Zn superoxide dismutase (SOD) are easily expressed in Escherichia coli, the amyotrophic lateral sclerosis-associated mutants have a strong propensity to aggregate into inclusion bodies. The alanine to valine mutation at the fourth codon (A4V) is responsible for a rapidly progressive disease course and is particularly prone to aggregation when expressed in E. coli. We found that A4V SOD remained soluble when expressed at 18 degrees C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23 degrees C or above. The SOD aggregates dissolved with 4 M urea, suggesting that intermolecular hydrophobic interactions were predominantly responsible for making SOD insoluble. Many of the urea-solubilized subunits were cross-linked via disulfide bridges. Fully active mutant SOD could be produced by dialyzing urea away in the presence of beta-mercaptoethanol and subsequently adding copper plus zinc, providing a fast procedure for purifying hundreds of milligrams of protein. Extensive rinsing removed most contaminating E. coli proteins from A4V SOD inclusion bodies except for a 37 kDa protein identified as outer membrane protein F using MALDI ToF/ToF mass spectrometry. Our results indicate that metal-deficient ALS-mutant SOD folds into stable apo conformation able to rebind metals. At high protein concentrations, SOD forms aggregates through hydrophobic interactions between subunits that seem to act as a kinetic snare to entrap additional proteins.     

Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli

The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.