Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-forming Escherichia coli cells

Misfolding-prone proteins produced in bacteria usually fail to adopt their native conformation and aggregate. In cells producing folding-reluctant protein species, folding modulators are supposed to be limiting, a fact that enhances protein deposition. Therefore, coproducing DnaK or other main chaperones along with the target protein has been a common approach to gain solubility, although with very inconsistent and often discouraging results. In an attempt to understand the reason for this inconsistency, the impact of exogenous DnaK (encoded in an accompanying plasmid) on two protein features observed as indicators of protein quality, namely solubility and functionality, has been analysed here through the specific fluorescence emission of a reporter Green Fluorescent Protein (GFP). Intriguingly, GFP solubility is strongly dependent on its own yield but poorly affected by DnaK levels. On the contrary, the specific fluorescence of both soluble and insoluble GFP populations is simultaneously modulated by the availability of DnaK, with a profile that is clearly dissimilar to that shown by protein solubility. Therefore, solubility, not being coincident with the biological activity of the target protein, might not be a robust indicator of protein quality.     

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.     

Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system

The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.     

Plasmid amplification in Escherichia coli after temperature upshift is impaired by induction of recombinant protein synthesis

Production of recombinant proteins often interferes with the physiology of the host organism by causing stress responses. In recombinant Escherichia coli, the cellular content of ColE1-derived plasmids and, consequently, the synthesis of the constitutively synthesized plasmid-encoded proteins generally increases after a temperature upshift. Simultaneous induction of inducible recombinant proteins that are synthesized at high levels and tend to form inclusion bodies, however, attenuates the plasmid amplification. This phenomenon was observed using temperature- as well as IPTG-inducible expression systems. Thus, high-level recombinant gene expression in connection with inclusion body formation does not only interfere with host cell but also with plasmid-related functions.     

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

Endoplasmic reticulum quality control and the unfolded protein response: insights from plants

Protein quality control (QC) within the endoplasmic reticulum and the related unfolded protein response (UPR) pathway of signal transduction are major regulators of the secretory pathway, which is involved in virtually any aspect of development and reproduction. The study of plant-specific processes such as pathogen response, seed development and the synthesis of seed storage proteins and of particular toxins is providing novel insights, with potential implications for the general recognition events and mechanisms of action of QC and UPR.     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.     

Effect of temperature on protein quality in bacterial inclusion bodies

Increasing evidence indicates that protein aggregation in bacteria does not necessarily imply loss of biological activity. Here, we have investigated the effect of growth-temperature on both the activity and stability of the inclusion bodies formed by a point-mutant of Abeta42 Alzheimer peptide, using green fluorescent protein as a reporter. The activity in the aggregates inversely correlates with the temperature. In contrast, inclusion bodies become more stable in front of chemical denaturation and proteolysis when temperature increases. Overall, the data herein open new perspectives in protein production, while suggesting a kinetic competition between protein folding and aggregation during recombinant protein expression.     

Protein folding, misfolding, and refolding of therapeutic proteins

Substantial progress has been made towards understanding the folding mechanisms of proteins in vitro and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible clues for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the past decade are summarized.     

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 μg/mL in the control to 40 μg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 μg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.     

The impact of E. coli O157 on the food industry

The relatively recent emergence of Escherichia coli O157 as a foodborne pathogen has had a significant impact on the food industry. This serovar possesses a number of undesirable characteristics that combine to make it one of the most serious threats to food safety in recent years. The widespread and sporadic occurrence in the gastrointestinal tracts of cattle, sheep, man and many other species, sometimes in the absence of any disease, and the complex nature of its ecology mean that it is not feasible to easily eradicate this serovar. In order to control and minimise foodborne disease by these organisms, it has been necessary to change primary production, processing, retailing and consumer-handling practices. Despite these new control measures, foodborne disease outbreaks caused by these microorganisms continue to occur and are accompanied by an increase in the number of cases of illness in many countries. This revised version was published online in November 2006 with corrections to the Cover Date.     

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S² constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.     

Quantitative and spatio‐temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re‐localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re‐targeted to alternative sites such as the inner membrane in the presence of site‐specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage‐free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.     

A 70-kDa molecular chaperone,DnaK, from the industrial bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: gene cloning,purification and molecular characterization of the recombinant protein

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a V _max of 32.5 nmol Pi/min and a K _M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg²⁺ and K⁺ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.     

The Effect of Pyrophosphate Analogues on the Activity of the Inorganic Pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> heat shock protein 70 is able to suppress the thermosensitivity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> DnaK mutant strain

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (dnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery.