Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli.

Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.     

Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli

Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.     

Routes to active proteins from transformed microorganisms.

Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.     

Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli

The enteric bacterium Escherichia coli is the most extensively used prokaryotic organism for production of proteins of therapeutic or commercial interest. However, it is common that heterologous over-expressed recombinant proteins fail to properly fold resulting in formation of insoluble aggregates known as inclusion bodies. Complex systems have been developed that employ simultaneous over-expression of chaperone proteins to aid proper folding and solubility during bacterial expression. Here we describe a simple method whereby a protein of interest, when fused in frame to the E. coli chaperones DnaK or GroEL, is readily expressed in large amounts in a soluble form. This system was tested using expression of the mouse prion protein PrP, which is normally insoluble in bacteria. We show that while in trans over-expression of the chaperone DnaK failed to alter partitioning of PrP from the insoluble inclusion body fraction to the soluble cytosol, expression of a DnaK–PrP fusion protein yielded large amounts of soluble protein. Similar results were achieved with a fragment of insoluble Varicella Zoster virus protein ORF21p. In theory this approach could be applied to any protein that partitions with inclusion bodies to render it soluble for production in E. coli.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.     

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.     

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.     

Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

Selective expression of the soluble product fraction in Escherichia coli cultures employed in recombinant protein production processes

Recombinant proteins produced in Escherichia coli hosts may appear within the cells’ cytoplasm in form of insoluble inclusion bodies (IB’s) and/or as dissolved functional protein molecules. If no efficient refolding procedure is available, one is interested in obtaining as much product as possible in its soluble form. Here, we present a process engineering approach to maximizing the soluble target protein fraction. For that purpose, a dynamic process model was developed. Its essential kinetic component, the specific soluble product formation rate, if represented as a function of the specific growth rate and the culture temperature, depicts a clear maximum. Based on the dynamic model, optimal specific growth rate and temperature profiles for the fed-batch fermentation were determined. In the course of the study reported, the mass of desired soluble protein was increased by about 25%. At the same time, the formation of inclusion bodies was essentially avoided. As the optimal cultivation procedure is rather susceptible to distortions, control measures are necessary to guarantee that the real process can be kept on its desired path. This was possible with robust closed loop control. Experimental process validation revealed that, in this way, high dissolved product fractions could be obtained at an excellent batch-to-batch reproducibility.     

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.     

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.     

Role of molecular chaperones in inclusion body formation

Protein misfolding and aggregation are linked to several degenerative diseases and are responsible for the formation of bacterial inclusion bodies. Roles of molecular chaperones in promoting protein deposition have been speculated but not proven in vivo. We have investigated the involvement of individual chaperones in inclusion body formation by producing the misfolding-prone but partially soluble VP1LAC protein in chaperone null bacterial strains. Unexpectedly, the absence of a functional GroEL significantly reduced aggregation and favoured the incidence of the soluble protein form, from 4 to 35% of the total VP1LAC protein. On the other hand, no regular inclusion bodies were then formed but more abundant small aggregates up to 0.05 microm(3). Contrarily, in a DnaK(-) background, the amount of inclusion body protein was 2.5-fold higher than in the wild-type strain and the average volume of the inclusion bodies increased from 0.25 to 0.38 microm(3). Also in the absence of DnaK, the minor fraction of soluble protein appears as highly proteolytically stable, suggesting an inverse connection between proteolysis and aggregation managed by this chaperone. In summary, GroEL and DnaK appear as major antagonist controllers of inclusion body formation by promoting and preventing, respectively, the aggregation of misfolded polypeptides. GroEL might have, in addition, a key role in driving the protein transit from the soluble to the insoluble cell fraction and also in the opposite direction. Although chaperones ClpB, ClpA, IbpA and IbpB also participate in these processes, the impact of the respective null mutations on bacterial inclusion body formation is much more moderate.     

A support vector machine-based method for predicting the propensity of a protein to be soluble or to form inclusion body on overexpression in Escherichia coli

MOTIVATION: Inclusion body formation has been a major deterrent for overexpression studies since a large number of proteins form insoluble inclusion bodies when overexpressed in Escherichia coli. The formation of inclusion bodies is known to be an outcome of improper protein folding; thus the composition and arrangement of amino acids in the proteins would be a major influencing factor in deciding its aggregation propensity. There is a significant need for a prediction algorithm that would enable the rational identification of both mutants and also the ideal protein candidates for mutations that would confer higher solubility-on-overexpression instead of the presently used trial-and-error procedures. RESULTS: Six physicochemical properties together with residue and dipeptide-compositions have been used to develop a support vector machine-based classifier to predict the overexpression status in E.coli. The prediction accuracy is approximately 72% suggesting that it performs reasonably well in predicting the propensity of a protein to be soluble or to form inclusion bodies. The algorithm could also correctly predict the change in solubility for most of the point mutations reported in literature. This algorithm can be a useful tool in screening protein libraries to identify soluble variants of proteins.     

Presence of the propeptide on recombinant lysosomal dipeptidase controls both activation and dimerization

Lysosomal dipeptidase catalyzes the hydrolysis of dipeptides with unsubstituted terminals. It is a homodimer and binds zinc. Dimerization is an important issue in understanding the enzyme's function. In this study, we investigated the influence of the propeptide on the folding and dimerization of recombinant lysosomal dipeptidase. For this purpose, we separately cloned and overexpressed the mature protein and the proenzyme. The overexpressed proteins were localized exclusively to insoluble inclusion bodies. Refolding of the urea-solubilized inclusion bodies showed that only dipeptidase lacking the propeptide was dimeric. The soluble renatured proenzyme was a monomer, although circular dichroism and fluorescence spectra of the proenzyme indicated the formation of secondary and tertiary structure. The propeptide thus controls dimerization, as well as activation, of lysosomal dipeptidase.     

Cloning and expression of the gene of chymotrypsin-like protease of human kallikrein-7 in Escherichia coli and isolation of recombinant protein

A sample of the human cDNA was used to amplify the segment encoding biosynthesis of chymotrypsin-like protease of kallikrein-7 and its cloning into the expressing plasmid pET23a(+).Biosynthesis of KLK-7 in transformed E. coil BL21(DE3) cells was accompanied by formation of insoluble inclusion bodies. The recombinant KLK-7 was extracted from the inclusion bodies using 7 M urea in the presence of 2-mercaptoethanol. The extracted recombinant KLK-7 was purified using methods of metal-chelate and ion-exchange chromatography, converted into a soluble form, and used for preparing monospecific antiserum.     

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.     

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.     

Amyloid-like properties of bacterial inclusion bodies

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.