Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMB 9871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111 units g^–1 cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone.     

Complementation studies of the DnaK–DnaJ–GrpE chaperone machineries from <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>, both in vivo and in vitro

The marine bacterium Vibrio harveyi is a potential indicator organism for evaluating marine environmental pollution. The DnaK–DnaJ–GrpE chaperone machinery of V. harveyi has been studied as a model of response to stress conditions and compared to the Escherichia coli DnaK system. The genes encoding DnaK, DnaJ and GrpE of V. harveyi were cloned into expression vectors and grpE was sequenced. It was found that V. harveyi possesses a unique organization of the hsp gene cluster (grpE–gltP–dnaK–dnaJ), which is present exclusively in marine Vibrio species. In vivo experiments showed that suppression of the E. coli dnaK mutation by V. harveyi DnaK protein was weak or absent, while suppression of the dnaJ and grpE mutations by V. harveyi DnaJ and GrpE proteins was efficient. These results suggest higher species-specificity of the DnaK chaperone than the GrpE and DnaJ cochaperones. Proteins of the DnaK chaperone machinery of V. harveyi were purified to homogeneity and their efficient cooperation with the E. coli chaperones in the luciferase refolding reaction and in stimulation of DnaK ATPase activity was demonstrated. Compared to the E. coli system, the purified DnaK–DnaJ–GrpE system of V. harveyi exhibited about 20% lower chaperoning activity in the luciferase reactivation assay. ATPase activity of V. harveyi DnaK protein was at least twofold higher than that of the E. coli model DnaK but its stimulation by the cochaperones DnaJ and GrpE was significantly (10 times) weaker. These results indicate that, despite their high structural identity (approximately 80%) and similar mechanisms of action, the DnaK chaperones of closely related V. harveyi and E.coli bacteria differ functionally.     

Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins

Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2–2.3-fold by coexpression of GroEL/ES, Trx, or DsbB–DsbD (DsbBD), while it was decreased by coexpression of DnaK–DnaJ–GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli.     

Chaperone over-expression in Escherichia coli: Apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates

The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.     

Cloning,expression, and characterization of a Baeyer–Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 50106 in <Emphasis Type="Italic">E. coli</Emphasis>

A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Characterization of catabolite repression and the promoter of the alginate lyase gene (aly) from Pseudomonas sp.

Summary Expression of the alginate lyase gene (aly) from Pseudomonas sp. W7 in Escherichia coli was very high (about 40%) and regulated by catabolite repression. The aly promoter was independent upon activation by cyclic AMP-cAMP receptor protein (cAMP-CRP) complex. When the aly promoter was removed by the construction of pKAL22, alginate lyase, which was under the control of lac promoter in E.coli, was markedly decreased (about 10%). This result confirmed that the aly promoter has stronger expression system in E.coli than lac promoter.     

Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein

The Escherichia coli molecular chaperone protein ClpB is a member of the highly conserved Hsp100/Clp protein family. Previous studies have shown that the ClpB protein is needed for bacterial thermotolerance. Purified ClpB protein has been shown to reactivate chemically and heat-denatured proteins. In this work we demonstrate that the combined action of ClpB and the DnaK, DnaJ, and GrpE chaperones leads to the activation of DNA replication of the broad-host-range plasmid RK2. In contrast, ClpB is not needed for the activation of the oriC-dependent replication of E. coli. Using purified protein components we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the plasmid RK2 replication initiation protein TrfA by converting inactive dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1, the corresponding protein system from yeast mitochondria, cannot activate the TrfA replication protein. Our results demonstrate for the first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein monomerization and in the activation of a DNA replication factor.     

ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli.

ClpB is a heat-shock protein from Escherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.     

In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli

In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.     

Successive action of Escherichia coli chaperones in vivo

Escherichia coli DnaK, DnaJ and GrpE are required for renaturation of heat-inactivated λ CI857 repressor (Gaitanaris et al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full-length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.     

Overproduction of d-hydantoinase and carbamoylase in a soluble form in Escherichia coli

The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli. Received: 26 November 1999 / Received revision: 28 February 2000 / Accepted: 10 March 2000     

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the detection of B-type natriuretic peptide

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of l-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with l-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration (1.0 mM l-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.     

Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.     

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.     

The Proline-rich Antibacterial Peptide Bac7 Binds to and Inhibits in vitro the Molecular Chaperone DnaK

Bac7, a cathelicidin peptide of the proline-rich group, inactivates bacteria in a stereospecific manner by entering target cells without any apparent membrane damage and by binding to as yet unknown intracellular targets. The present study was aimed at detecting these putative intracellular interactors, which might mediate the antibacterial action of this peptide. By using affinity resins functionalized with the N-terminal 1-35 fragment of Bac7, a single protein was specifically retained with high affinity from Escherichia coli cytoplasmic protein lysates. This ligand was identified as the heat shock protein DnaK, the Hsp70 homolog in E. coli. The interaction between the peptide and the chaperone is stereospecific, given that a resin prepared with the all- d enantiomer failed to retain the protein. In vitro, Bac7(1-35) formed a complex with DnaK with an affinity comparable to that of other known high-affinity peptide ligands. In addition, at 10–100 μM concentration, the peptide inhibited the protein refolding activity of the complete DnaK/DnaJ/GrpE/ATP molecular chaperone system in a dose-dependent manner. Despite these results, the in vitro sensitivity to the peptide, under growth permitting conditions, of DnaK-deficient E. coli strains was not significantly affected compared to the wild-type strain. This suggests that, apart from DnaK, other vital targets for the proline-rich AMPs are present in susceptible bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Marco Scocchi and Christine Lüthy contributed equally to this work.     

DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.