Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria

Time-dependent aggregation of a plasmid-encoded β-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 μm³ h⁻¹, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.     

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.     

The impact of dnaKJ overexpression on recombinant protein solubility results from antagonistic effects on the control of protein quality

We have produced increasing levels of DnaK and its co-chaperone DnaJ along with the model VP1LAC misfolding-prone protein, to explore the role of DnaK on the management of Escherichia coli inclusion bodies. While relative solubility of VP1LAC is progressively enhanced, the heat-shock response is down-regulated as revealed by decreasing levels of GroEL. This is accompanied by an increasing yield of VP1LAC and a non-regular evolution of its insoluble fraction, at moderate levels of DnaK resulting in more abundant inclusion bodies. Also, the impact of chaperone co-expression is much more pronounced in wild type cells than in a DnaK- mutant, probably due to the different background of heat shock proteins in these cells. The involvement of DnaK in the supervision of misfolding proteins is then pictured as a dynamic balance between its immediate holding and folding activities, and the side-effect downregulation of the heat shock response though the limitation of other chaperone and proteases activities.     

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.     

Refolding process of cysteine-rich proteins:Chitinase as a model

Background:

Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.

Methods:

Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.

Results:

After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.

Conclusions:

By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.Key Words: Chitinase, Cysteine-rich proteins, Protein refolding, Protein solubilization     

Improving the soluble expression of aequorin in Escherichia coli using the chaperone-based approach by co-expression with artemin

Abstract

Escherichia coli is a common host that is widely used for producing recombinant proteins. However, it is a simple approach for production of heterologous proteins; the major drawbacks in using this organism include incorrect protein folding and formation of disordered aggregated proteins as inclusion bodies. Co-expression of target proteins with certain molecular chaperones is a rational approach for this problem. Aequorin is a calcium-activated photoprotein that is often prone to form insoluble inclusion bodies when overexpressed in E. coli cells resulting in low active yields. Therefore, in the present research, our main aim is to increase the soluble yield of aequorin as a model protein and minimize its inclusion body content in the bacterial cells. We have applied the chaperone-assisted protein folding strategy for enhancing the yield of properly folded protein with the assistance of artemin as an efficient molecular chaperone. The results here indicated that the content of the soluble form of aequorin was increased when it was co-expressed with artemin. Moreover, in the co-expressing cells, the bioluminescence activity was higher than the control sample. We presume that this method might be a potential tool to promote the solubility of other aggregation-prone proteins in bacterial cells.     

Affinity purification of antibodies by using Ni2+-resins on which inclusion body-forming proteins are immobilized

Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound.     

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.     

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.     

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.     

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.     

重组抗血红素ScFv包涵体蛋白的复性及纯化研究

抗血红素多二硫键ScFv在大肠杆菌中绝大多数表达产物为包涵体,为了获得可溶性的具有生物活性的ScFv,摸索了不同的复性条件,包括透析法、稀释和层析相结合的方法。研究发现,先对溶解的变性ScFv溶液稀释,进行初步的蛋白质复性,再利用Sephadex G-25凝胶层析进一步复性、降低变性剂浓度和纯化,至少可以得到95％纯度,产率为150mg／L的目标蛋白,通过一次凝胶过滤层析,达到了去除变性剂、复性及纯化ScFv蛋白三种目的,为多二硫键ScFv在大肠杆菌中的表达和纯化提供了一种经济可行的方法。     

Folding of a misfolding-prone beta-galactosidase in absence of DnaK

In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model beta-galactosidase fusion protein VP1LAC produced in DnaK(-) cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK(+) cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK(-) background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli beta-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK.     

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.     

Learning about protein solubility from bacterial inclusion bodies

The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed.     

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.     

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.     

Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology

Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of inclusion bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques.     

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.