A New Artificial Chaperone for Protein Refolding: Sequential Use of Detergent and Alginate

A novel artificial chaperone system using a combination of detergents and alginate was developed to refold three enzymes with totally different structures. Upon dilution of denatured protein in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used alginate, a linear copolymer of d-mannuronic acid and l-guluronic acid, to initiate and complete the refolding process. The results indicated that the extent of refolding assistance for the proteins was different due to detergent structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic and hydrophobic interactions among the capturing and stripping agents used in this investigation. Based on this newly developed method, it is expected that the protein refolding operation can be achieved easily, cheaply and efficiently.     

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C₁₂E₈). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C₁₂E₈ leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C₁₂E₈ were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C₁₂E₈ recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C₁₂E₈ was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.     

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9_β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9_α + β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9_β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9_α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.     

Chaperones Rescue Luciferase Folding by Separating Its Domains

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Alkaline phosphatase refolding assisted by sequential use of oppositely charged detergents: a new artificial chaperone system

A novel artificial chaperone system, based on combination of oppositely charged detergents, was elaborated to refold soluble alkaline phosphatase. Upon dilution of urea-denatured alkaline phosphatase to a nondenaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used detergents with variable charges and tail lengths to initiate and complete the refolding process. The results obtained from various analyses (fluorescence, UV, circular dichroism, surface tension, turbidity measurements and activity assays) indicated that the extent of refolding assistance was different due to detergents structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic interactions among the capturing and stripping detergents used in this investigation. Collectively it is expected that protein refolding process can be achieved easier, cheaper and more efficient, using the new technique reported here.     

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.     

Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A₂ (sPLA₂s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A₂ (hsPLA₂-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA₂-IID in Escherichia coli, the DNA-coding sequence for hsPLA₂-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA₂-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A₂. The refolded recombinant hsPLA₂-IID demonstrated Ca²⁺-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA₂-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.     

Do nucleic acids moonlight as molecular chaperones?

Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation.     

Oxidative refolding of lysozyme assisted by DsbA, DsbC and the GroEL apical domain immobilized in cellulose

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBD_Cex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r² > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k _N) and aggregation (k _A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.     

High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system

The overexpression of subunit b of F₁F₀ adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F₁F₀ ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.     

GroEL的耗能分子伴侣机制

双环结构Gro EL及其辅分子伴侣Gro ES是目前研究得最深入的分子伴侣.然而,Gro EL/Gro ES帮助蛋白质折叠的一些关键理化机制,尤其是水解ATP,Gro EL发生构象改变,能否主动调节蛋白质错误折叠中间体的构象,以促进错误折叠中间体的复性,仍然存在争议.结合本研究组近年的工作,作者着力介绍Gro EL促进蛋白质折叠的主动解折叠机制.     

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2(271-326))

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2_271–326, a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2_271–326, was subsequently purified by reverse-phase HPLC and confirmed by SDS–PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2_271–326 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.     

Comparative Evaluation of α-Amylase Refolding Through Two Different Artificial Chaperone Systems

Two different artificial chaperone systems were evaluated in this work using either detergents or CDs as the stripping agents. Upon dilution of urea-denatured α-amylase to a non-denaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to remove the detergent molecules. Our results by fluorescence, UV, turbidity measurement, circular dichroism, surface tension and activity assay indicated that the extent of refolding assistance was different due to different inter- and intra- molecular interactions in the two different systems. However, the high activity recovery in the presence of detergents, as the stripping agent, suggests that they can constitute suitable replacement for the more expensive and common stripping agent of cyclodextrins.     

Problems in Obtaining Diffraction-quality Crystals of Hetero-oligomeric Integral Membrane Proteins

The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein–protein contacts presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome b₆f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein–protein contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of colicin E3, the latter served as a ligand for protein–protein contacts that facilitated crystallization.     

Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1

G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the β1 and β2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli.     

In vitro Evolution of Uracil Glycosylase Towards DnaKJ and GroEL Binding Evolves Different Misfolded States

Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (?U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB.     

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.     

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N‐ and C‐terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast‐localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N‐terminal arms located on the outside of the dodecamer and six inwardly‐facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small‐angle X‐ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone‐client complexes were monitored and the R_g‐values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N‐terminal arms of the Hsp21 dodecamer. No such increase in R_g‐values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone‐client interactions involve the N‐terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N‐terminal arms of Hsp21 can trap and refold early‐unfolding intermediates with or without dodecamer dissociation.