Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.     

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.     

Strategies for folding of affinity tagged proteins using GroEL and osmolytes

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.     

Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli

Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.     

Only the reduced conformer of alpha-lactalbumin is inducible to aggregation by protein aggregates

Reduced apo-alpha-lactalbumin (r-LA) in the pre-molten globule state is soluble in neutral and reduced buffer at 25 degrees C but becomes aggregated when aggregates of various proteins are added. However, protein aggregates do not induce the aggregation of apo-alpha-lactalbumin in the molten globule state. The presence of the molecular chaperone protein disulfide isomerase or the "chemical chaperone" polyethyleneglycol inhibits the induced aggregation. Native proteins, aggregation-free folding intermediates, and soluble aggregates do not induce the aggregation. The interaction between r-LA and protein aggregates is hydrophobic in nature. These findings suggest that pre-molten globule state of LA is the target not only for chaperones but also for protein aggregates.     

Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli

During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.     

Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli

Eight combinations of molecular chaperones (e.g., DnaK/DnaJ/GrpE/ClpB) are co-expressed with the target recombinant protein to compare their effectiveness in improving its soluble yield. This system allows the most complete and rational approach proposed so far to use the chaperone activity for optimizing the host cell folding machinery. Furthermore, a two-step protocol is presented, in which protein synthesis and protein refolding are uncoupled. Molecular chaperones and target protein accumulate in the first growth phase and target protein aggregates are then disaggregated in vivo after the block of protein synthesis. The optimal chaperone combination to maximize the soluble yield of a specific protein remains unpredictable. Therefore, a small-scale purification selection step is useful for screening among expression combinations before scaling-up production. Applying such a strategy, we could increase the solubility of 70% of the tested constructs with yields of up to 42-fold more protein than in controls. The procedure takes 2 d.     

Chaperones in control of protein disaggregation

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.     

Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli

Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V(H)) and light chain (V(L)) cloned from the hybridoma 3G7 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-DON V(H) was a member of the V(H) III gene family IA subgroup and the V(L) gene belonged to the Vlambda gene family II subgroup. Extensive efforts to express the functional scFv antibody in E. coli have been made by using gene fusion and chaperone coexpression. Coexpression of the molecular chaperones (DnaK-DnaJ-GrpE) allowed soluble expression of the scFv. The scFv antibody fused with hexahistidine residues at the C-terminus was purified by immobilized metal affinity chromatography (IMAC). Soluble scFv antibody produced in this manner was characterized for its antigen-binding characteristics. Its biological affinity as antibody was measured by surface plasmon resonance (SPR) analysis and proved to be significant but weaker than that of the whole anti-DON mAb.     

Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones

Molecular chaperones are essential for the correct folding of proteins in the cell under physiological and stress conditions. Two activities have been traditionally attributed to molecular chaperones: (1) preventing aggregation of unfolded polypeptides and (2) assisting in the correct refolding of chaperone-bound denatured polypeptides. We discuss here a novel function of molecular chaperones: catalytic solubilization and refolding of stable protein aggregates. In Escherichia coli, disaggregation is carried out by a network of ATPase chaperones consisting of a DnaK core, assisted by the cochaperones DnaJ, GrpE, ClpB, and GroEL-GroES. We suggest a sequential mechanism in which (a) ClpB exposes new DnaK-binding sites on the surface of the stable protein aggregates; (b) DnaK binds the aggregate surfaces and, by doing so, melts the incorrect hydrophobic associations between aggregated polypeptides; (c) ATP hydrolysis and DnaK release allow local intramolecular refolding of native domains, leading to a gradual weakening of improper intermolecular links; (d) DnaK and GroEL complete refolding of solubilized polypeptide chains into native proteins. Thus, active disaggregation by the chaperone network can serve as a central cellular tool for the recovery of native proteins from stress-induced aggregates and actively remove disease-causing toxic aggregates, such as polyglutamine-rich proteins, amyloid plaques, and prions.     

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.     

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.     

Protein solubility and folding enhancement by interaction with RNA

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.     

From the cradle to the grave: molecular chaperones that may choose between folding and degradation

Molecular chaperones are known to facilitate cellular protein folding. They bind non-native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy-dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.     

Folding and refolding of proteins in chromatographic beds

The correct folding of solubilized recombinant proteins is of key importance for their production in industry. On-column refolding of proteins is mainly achieved by three methods: size-exclusion chromatography, ion exchange chromatography and affinity chromatography using immobilized metal chelates. The principles of these methods were first laid down in the 1990s, but many recent improvements have been made to these processes including sophisticated changes to the mobile phase composition and the recycling of aggregates to improve yield. Advances have also been made in the use of immobilized metal affinity chromatography and by mimicking the natural folding process with artificial chaperones.     

Use of folding modulators to improve heterologous protein production in Escherichia coli

Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed – and largely unpredictable – results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies.     

The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.     

Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs

A great deal of attention has been paid to so-called amyloid diseases, in which the proteins responsible for the cell death and resultant diseases undergo conformational changes and aggregate in vivo, although whether aggregate formation is the cause or the result of the cell death is controversial. Recently, an increasing attention is given to protein folding diseases tightly associated with mutations. These mutations result in temperature-dependent misfolding and hence inactivation of the proteins, leading to loss of function, at physiological temperature; at low so-called permissive temperatures, the mutant proteins correctly fold and acquire functional structure. Alternatively, activation can be induced by use of osmolytes, which restores the folding of the mutant proteins and hence are called chemical chaperones. The osmolytes are compatible with macromolecular function and do stabilize the native protein structure. However, chemical chaperones require high concentrations for effective folding of mutant proteins and hence are too toxic in in-vivo applications. This limitation can be overcome by pharmacological chaperones, whose functions are similar to the chemical chaperones, but occur at much lower concentrations, i.e., physiologically acceptable concentrations. Although the research and clinical importance of pharmacological chaperones has been emphasized, the initial and central concept of osmolytes is largely ignored. Here we attempt to bridge the concept of osmolytes to applications of pharmacological chaperones.     

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter⁻¹), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter⁻¹) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.