Review
`
`TRENDS in Biotechnology Vol.24 No.4 April 2006
`
`Protein quality in bacterial
`inclusion bodies
`Salvador Ventura1,3 and Antonio Villaverde1,2
`
`1Institut de Biotecnologia i de Biomedicina, Universitat Auto` noma de Barcelona, Bellaterra, 08193 Barcelona, Spain
`2Departament de Gene` tica i de Microbiologia, Universitat Auto` noma de Barcelona, Bellaterra, 08193 Barcelona, Spain
`3Departament de Bioquı´mica i de Biologia Molecular, Universitat Auto` noma de Barcelona, Bellaterra, 08193 Barcelona, Spain
`
`A common limitation of recombinant protein production
`in bacteria is the formation of
`insoluble protein
`aggregates known as inclusion bodies. The propensity
`of a given protein to aggregate is unpredictable, and the
`goal of a properly folded, soluble species has been
`pursued using four main approaches: modification of
`the protein sequence;
`increasing the availability of
`folding assistant proteins; increasing the performance
`of the translation machinery; and minimizing physico-
`chemical conditions favoring conformational stress and
`aggregation. From a molecular point of view, inclusion
`bodies are considered to be formed by unspecific
`hydrophobic interactions between disorderly deposited
`polypeptides, and are observed as ‘molecular dust-balls’
`in productive cells. However, recent data suggest that
`these protein aggregates might be a reservoir of
`alternative conformational states, their formation
`being no less specific than the acquisition of the
`native-state structure.
`
`Introduction
`Recombinant protein production is an essential tool for the
`biotechnology industry and also supports expanding areas
`of basic and biomedical research, including structural
`genomics and proteomics. Although bacteria still rep-
`resent a convenient production system, many recombi-
`nant polypeptides produced in prokaryotic hosts undergo
`irregular or incomplete folding processes that usually
`result in their accumulation as insoluble, and usually
`refractile, aggregates known as inclusion bodies (IBs)
`[1,2]. In fact, the solubility of bacterially produced proteins
`is of major concern in production processes [3,4] because
`IBs are commonly formed during overexpression of
`heterologous genes, particularly of mammalian or viral
`origin. Consequently, many biologically relevant protein
`species are excluded from the market because they cannot
`be harvested in the native form at economically con-
`venient yields. Although some recombinant proteins do
`occur in both the soluble and insoluble cell fractions, many
`others are only produced as IBs. To date, the solubility of a
`given gene product has not been anticipated before gene
`expression. However, it is now clear that the extent of
`protein aggregation is determined, at least partially, by a
`combination of process parameters,
`including culture
`
`Corresponding author: Villaverde, A. (avillaverde@servet.uab.es).
`Available online 28 February 2006
`
`media composition, growth temperature, production rate
`(as result of diverse factors, such as gene dosage, promoter
`strength, mRNA stability and codon usage) [5,6], and the
`availability of heat-shock chaperones [7,8]. All of these
`factors can be manipulated to enhance solubility but the
`operational range is more limited than that required for a
`competent solubility control. Overexpression of chaper-
`ones and other folding modulators along with the
`recombinant gene has been the most successful approach
`for the minimization of IB formation. During the past
`decade, hundreds of articles have described particular
`chaperone-assisted production experiments with poorly
`concluding results, often because of inconsistencies when
`considering different protein species, host cell strains or
`expression systems [8,9]. Although still a matter of
`speculation, the origin of such variability might lie in
`the distinct requirements of different proteins when
`folding in a prokaryotic environment.
`In addition, despite the functional redundancy of the
`quality control system, the activities of some chaperones
`(such as DnaK) cannot be completely complemented by
`others [10], and their titration causes bottlenecks in the
`folding process [11]. It is also true that an important part
`of the bacterial protein quality-control system is organized
`into partially overlapping sequential networks, in which
`folding intermediates are delivered from one chaperone
`(or chaperone set) to another [12,13]. This sequential
`handling would prevent the proper folding of a misfolding-
`prone species when one crucial folding element is not
`available at the required concentrations; however, the
`overexpression of this bottleneck chaperone would make
`the next step of the folding process limiting.
`Alternatively, IBs can be a source of relatively pure
`protein because they can be easily purified from disrupted
`cells. By using IBs as a starting material, and after
`applying in vitro refolding procedures, native proteins can
`be recovered ready for use [14–20]. The main concern
`about using IBs as a source material for industrial
`purposes is that in vitro refolding procedures are not
`universal and need to be adapted for each specific protein.
`In addition, the cost and speed of such refolding
`procedures are not always convenient in the large-scale
`formats needed in industry [15,21].
`The undesired aggregation of recombinant proteins has
`been experienced since early recombinant DNA technol-
`ogies were developed. However, the physiological and
`
`www.sciencedirect.com 0167-7799/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2006.02.007
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`Review
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`TRENDS in Biotechnology Vol.24 No.4 April 2006
`
`structural data that has been collected about IBs during
`the past five years are now offering the first steps towards
`an integrated model of protein aggregation in bacteria
`[22]. In addition, picturing how IB formation is connected
`to the physiology of the cell during the conformational
`stress imposed by protein overproduction is now
`becoming possible.
`
`Morphology and composition
`In actively producing recombinant E. coli cells, IBs are
`seen as refractile particles, usually occurring in the
`cytoplasm [23,24], although secretory proteins can also
`form IBs in the periplasm [25]. Under electron microscopy,
`IBs appear rather amorphous [26] but, after detergent-
`based purification, scanning microscopy reveals them to
`be rod-shaped particles [24,27]. In vitro protease digestion
`of purified inclusion bodies occurs on IB-associated
`proteins as a cascade process [28,29] in which target
`sites are sequentially activated or exposed to the enzyme
`in a defined manner. This in-order cleavage indicates both
`conformational flexibility and accessibility of IB proteins.
`Also, partially digested IBs have a granular architecture
`[27] that might be compatible with IBs being formed by
`the clustering of protease-resistant, smaller aggregates.
`Classical proteomics of IBs showed them to be relatively
`homogeneous in composition and mainly formed by the
`recombinant protein itself [30–32]. Although occurring in
`variable proportions, the recombinant product can reach
`more than 90% of the total embedded polypeptides [2,22],
`which is a convenient protein supply for further in vitro
`refolding. The remaining material includes proteolytic
`fragments of the recombinant protein [33,34], traces of
`membrane proteins [30,35], phospholipids and nucleic
`acids [31], at least some of these being contaminants
`retained during the IB purification procedures [36]. In E.
`coli IBs, the small heat-shock proteins IbpA and IbpB
`have been identified [22,37,38] in addition to the main
`chaperones DnaK and GroEL [22,35].
`
`Molecular determinants
`The large set of polypeptides forming bacterial IBs are not
`related, either structurally or sequentially, and include
`small,
`large, monomeric, multimeric, prokaryotic or
`eukaryotic proteins. Thus, aggregation inside bacterial
`factories has long been considered to be a nonspecific
`process, resulting in the formation of disordered intra-
`cellular precipitates. Accordingly, several general features
`inherent to the particular molecular status of the protein
`but irrespective of its nature have been suggested to
`promote IB formation. These include: high local concen-
`trations of the produced polypeptide; transient accumu-
`lation of proteins in totally or partially unfolded
`conformations, with reduced solubility related to that of
`the native form [3]; the accumulation of unstructured
`protein fragments as a result of proteolytic attack [19]; the
`establishment of wrong interactions with the bacterial
`folding machinery [39]; the lack of the post-translational
`modifications needed for the solubility of some eukaryotic
`polypeptides [40]; and the prevention of proper disulfide
`pairing in the reducing cytoplasmic environment [41].
`
`www.sciencedirect.com
`
`Although such environmental factors are relevant for
`IB formation, the intrinsic nature of a polypeptide and its
`sequence also determine its partitioning between the
`insoluble and soluble cell fractions. Several classical
`observations, together with recent results, reinforce this
`view. The high purity of the recombinant protein in IBs,
`and the recurrent observation that
`recombinant
`expression results in the formation of a reduced number
`of IBs (usually one) [23], suggest that they might be
`formed by the growth of a small number of initial founder
`aggregates by a nucleation-like mechanism relying on
`molecular recognition events. Several observations sup-
`port this view. First, specificity of polypeptide association
`during aggregation processes has been seen in in vitro
`refolding studies of proteins in complex protein mixtures
`[42]. Second, the folding intermediates of different
`proteins tend to self-associate, in vitro, instead of co-
`aggregating, despite the fact they form IBs when
`expressed individually in bacteria [43]. Finally, and more
`interestingly, under certain conditions, co-expression of
`two proteins from genes carried on the same plasmid
`results in the formation of two types of cytoplasmic
`aggregates, each enriched in one type of recombinant
`protein [44]. This segregation of the protein aggregates is
`not the result of a temporal dependence of deposition,
`supporting the view that, seeing as it occurs in vitro,
`aggregation of proteins into IBs is a selective process.
`IBs have long been thought to be devoid of all molecular
`architecture, according to the view that unspecific
`hydrophobic interactions drive the deposition process.
`However, pioneering studies in the early 1990s [45–47],
`together with more recent investigations [48–50], run
`against this view. The use of attenuated total reflectance
`infra-red spectroscopy for IBs analysis has shown that,
`irrespective of the native protein structure, formation of
`IBs results in the acquisition of significant new b-sheet
`structures compared with the native conformation, even
`for b-sheet-rich proteins. The persistence of some native
`conformation in addition to the presence of disordered
`chain segments has been also described, the content
`depending on the particular IB-forming protein [51]. The
`structural data suggest that the newly formed b-sheet
`architecture in IBs is stabilized by a network of hydrogen
`bonds between different chains, resulting in tightly
`packed,
`extended intermolecular b-sheets. These
`b-sheet-rich polypeptides or polypeptide regions would
`be resistant to proteolysis, and it is enticing to propose
`that they might constitute the above mentioned multiple
`protease-resistant nuclei within IBs, whereas proteins or
`protein segments in native and specially disordered
`conformations would constitute the protease-sensitive
`part of IBs.
`In this context, an obvious question arises: how do
`specific interactions that occur during the nucleation
`process result in a more or less common structure for all
`IBs? Although only a few studies have addressed this topic
`for IBs, it has been a key issue in the closely related area of
`protein misfolding and aggregation into amyloid fibrils.
`Independent of the forming protein, all amyloid fibrils
`share a predominant b-sheet architecture [52]. This
`conformation, as in the case of IBs, is stabilized mostly
`
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`TRENDS in Biotechnology Vol.24 No.4 April 2006
`
`181
`
`by the establishment of non-covalent interactions between
`polypeptide backbones, which are common to all proteins
`[53]. For amyloids, it has been proven that the propen-
`sities of protein backbones to aggregate are sharply
`modulated by their amino acid sequences, with certain
`stretches acting as ‘hot spots’ from which aggregation can
`nucleate specifically [54–56]. This can be the case for IBs
`too. Recently, it has been shown that a preformed IB can
`act as an effective aggregation seed for the deposition of its
`partially folded soluble protein counterpart in a dose-
`dependent manner [49]. Moreover, the seeding process is
`highly specific because IBs promote the deposition of
`homologous but not heterologous polypeptides [49].
`Sequestering of homologous misfolded species into IBs
`might be a refined mechanism to reduce the potential
`toxicity of partially folded monomers or small oligomers
`[57], of which the solvent-exposed hydrophobic surfaces
`might interact, improperly, with a large number of cellular
`components and/or exhaust the in vivo folding machinery,
`thereby hampering the folding and function of the cell
`proteins. Thus, the establishment of specific interactions
`during aggregation might be a conserved strategy with a
`role in cellular protection, which seems to be the case in
`IB-forming recombinant bacteria [58]. In summary,
`protein aggregation as bacterial IBs and as amyloid fibrils
`shows more than one coincident trait (Table 1).
`
`Sequence determinants
`The impact of point mutations on IB formation in several
`protein systems also suggests that the primary structure
`of a polypeptide somehow determines its propensity to
`aggregate into IBs, whereby specific changes have a huge
`impact on solubility. However, to forecast the effect of
`sequence changes on the aggregation propensity in E. coli
`still constitutes a challenge because the structural and
`thermodynamic context in which they occur must be taken
`into account, and these parameters are not easily
`predictable.
`Furthermore,
`consistently
`identical
`mutations in different protein systems have been shown
`to result in dissimilar effects [59–63]. Nevertheless, the
`increasing number of structural genomic initiatives, and
`the concomitant need for soluble recombinant proteins,
`
`Table 1. Main functional and structural traits of bacterial
`inclusion bodies resembling those of amyloids
`Feature
`High purity of the aggregate
`Aggregation mainly from folding intermediates
`Sequence-specific aggregation
`Chaperon-modulated aggregation
`Seeding-driven aggregation
`Aggregation propensities strongly affected by point
`mutations
`Reduced aggregation by stabilization of the native
`structure
`Intermolecular, cross b-sheet organization or in
`general, enrichment of b structure
`Fibril-like organization (of soluble protein aggregates)
`Amyloid-tropic dye binding
`Enhanced proteolytic resistance (of a fraction of IB
`protein species)
`Protection from cytotoxicity
`
`Refs
`[23]
`[49,89]
`[43,49]
`[11,90]
`[49]
`[91–95]
`
`[96,97]
`
`[47,49]
`
`[86]
`[49]
`[27,28]
`
`[58]
`
`www.sciencedirect.com
`
`has pushed several attempts to predict IB formation
`directly from the primary structure [64] but still with
`inconsistent results. Among the intrinsic factors proposed
`to be related to the propensity of a polypeptide to be
`incorporated into IBs are: the size of the polypeptide; its
`phylogenetic origin; the protein family and/or fold; the
`charge average; the proportion of aliphatic residues; the
`in vivo half-life; the frequency of occurrence of certain
`dipeptides and tripeptides within the sequence; the
`proportion of residues with good b-sheet propensity; and
`the fraction of turn-forming residues. The reasons behind
`the discordance among approaches rely on the inherent
`difficulty of the addressed problem, namely aggregation
`propensity is the net result of several extrinsic and
`intrinsic factors and many of them are important to
`different extents depending on the protein and expression
`contexts [65]. In addition, it is clear that the solubility of
`recombinant heterologous proteins has nothing to do with
`the forces that have shaped sequences during evolution.
`Thus, it is implausible that particular polypeptide proper-
`ties, which lead to increased solubility of a recombinant
`protein, would dominate in any given group of proteins.
`This hampers the detection of relevant patterns influen-
`cing IB formation.
`
`Protein quality and dynamics
`Overall, recent data suggests that IBs might embrace
`conformational states different to those observed in the
`soluble cell fraction, ranging from enriched b-forms to
`native or native-like structures [45,48–50] (Figure 1). The
`heterogeneous conformational status of IB protein was
`hinted by the modeling of in vitro IB proteolytic digestion,
`where different species with distinctive proteolytic sensi-
`tivity were detected [27,28]. Such heterogeneity is
`probably supported by the fact that the volumetric IB
`growth during gene overexpression is the result of
`unbalanced protein deposition and simultaneous cell-
`driven physiological removal. Interestingly, at least a
`fraction of IB protein is in continuous dynamic transition
`between soluble and insoluble cell fractions [33] and, in
`the absence of protein synthesis, cytoplasmic IBs are
`almost completely disintegrated in a few hours [66].
`Therefore, rather than being mere molecular ‘dust-balls’
`of the folding machinery, IBs are protein reservoirs that
`are profoundly integrated in the protein quality system of
`the cell
`[22], and the embedded protein is under
`continuous quality surveillance. Disaggregating ATPase-
`C
`), sharing conserved ATP
`associated chaperones (AAA
`binding and hydrolysis motifs (essentially ClpB), are
`probably key elements in IB protein release because
`they are responsible for protein reactivation in thermally
`stressed cells [67–70]. Small heat-shock proteins (IbpAB),
`commonly associated with IB proteins [38,71], are also
`important contributors to the disintegration process,
`acting in a chaperone team that includes ClpB and
`DnaK [72,73]. Other cytoplasmic chaperones, such as
`GroEL, GroES and ClpA, are probably assisting removal
`of the IB protein because, upon arrest of protein
`production, IBs are more stable in their respective absence
`[11,26]. Furthermore, in IB-forming recombinant E. coli
`cells, DnaK, GroEL and IbpAB have been identified as IB
`
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`TRENDS in Biotechnology Vol.24 No.4 April 2006
`
`Translational apparatus
`
`Refolding
`and Proteolysis
`
`Deposition
`
`Translational apparatus
`
`Soluble fraction
`
`Insoluble fraction
`
`TRENDS in Biotechnology
`
`Figure 1. Recombinant proteins produced in distant translational factories within
`the bacterial cytoplasm occur in either soluble or insoluble cell fractions. Such
`entities are virtual cell compartments (indicated by a vertical dashed line) between
`which proteins are distributed according to their fractionation under high-speed
`centrifugation. A fraction of de novo synthesized polypeptides can immediately
`reach the native conformation and are fully functional (yellow spheres). Other
`molecules enter into incorrect, dead-end folding pathways, are non functional and
`tend to aggregate because of the presence of solvent-exposed hydrophobic
`surfaces (small brown boxes). Aberrant folding forms and folding intermediates
`can have properly folded domains that, if embracing active sites, might be still fully
`or partially functional, although tending to aggregate (orange boxes). The
`backbones of these protein forms can interact in a sequence-dependent manner
`and under second-order kinetics to form small, b-sheet-enriched, soluble
`aggregates, organized as fibers or other cluster types. Soluble aggregates are
`trapped, specifically, in larger aggregation nuclei, forming one or a few IBs (vertical
`brown box in the insoluble cell fraction) according to first-order kinetics. Therefore,
`IBs contain both inactive (unfolded) and active (partially folded or eventually
`properly folded) protein species that might self-organize in a concentric manner.
`Here, native-like species surround unfolded, densely packaged and proteolytically
`stable polypeptide chains. Protein material is steadily transferred between these
`virtual cell compartments by either deposition into IBs or refolding and/or
`proteolysis of IB proteins, generating a conformational continuum between soluble
`and insoluble cell fractions. Therefore, incorrect folding and aggregation, or proper
`folding and solubility, are not perfectly pair-matched events because both active
`and inactive protein forms can be found in either the soluble or the insoluble
`fractions.
`
`components [22,35,38]. Intriguingly, most cellular DnaK
`molecules have been observed at the IB interface [26],
`where this chaperone probably acts by refolding or
`releasing IB polypeptides in cooperation with ClpB and
`IbpAB [67,72,74,75]. Recent insights on the disaggrega-
`tion process have provided fascinating details about its
`molecular mechanics. The protein ClpB recognizes sub-
`strates through the conserved Tyr251 residue sited at the
`central pore of the first AAA domain. This fact suggests a
`translocation event for ClpB-mediated protein removal
`[76,77] that acts on discrete protein molecules rather than
`on aggregated sections [78]. Both DnaK and ClpB middle
`domains might also contribute by providing an unfolding
`force in a still unsolved mechanism, acting in coordination
`with the translocation event [79].
`Conversely, it seems that proteases are secondary tools
`for aggregate processing, acting on IB polypeptides once
`released [66] or during disaggregation [80]; however,
`in situ digestion of IB protein has been suspected, through
`indirect in vivo and in vitro observations [23,28,80,81]. In
`support of a direct proteolytic attack, the absence of either
`Lon or ClpP proteases largely minimizes IB disintegration
`K
`background, IB proteins
`[82]. However,
`in a ClpP
`released to the soluble cell fraction remain stable and
`can refold to a functional form [82], highlighting this
`enzyme
`as
`a
`controller
`of
`the
`quality
`of
`disaggregated proteins.
`The heterogeneous conformational nature of IB pro-
`teins is,
`in addition, reflected by the relatively high
`activity of IBs formed by enzymes such as galactosidases
`and other glucanases [6,10,83] (Table 2). Recently, the
`same has been observed for aggregating fluorescent
`proteins that generate highly emitting IBs [84]. In fact,
`when analyzing the specific activity of soluble and IB
`forms of b-galactosidase fusions, such values are within
`the same order of magnitude [10]. This similarity can be
`partially attributed to the occurrence of ‘soluble aggre-
`gates’ [85], namely clusters of soluble but biologically
`inactive protein, organized as fibers, which might even-
`tually be among IB precursors [86]. Such elements would
`
`Table 2. Some structural and functional evidence that properly folded protein species are a significant component of bacterial IBs
`IB protein
`Structure (determination method)
`Biological activity (% relative to the
`Refs
`soluble counterpart, when determined)
`High IB fluorescence emission in vivo
`(between 20 and 30%)
`High specific activity in purified IBs
`(from around 30 up to more than 100%)
`Low activity in purified IBs (6%)
`High activity in purified IBs (25%)
`Detectable activity in purified IBs
`Detectable activity in purified IBs
`
`[84]
`
`[6,10,84]
`
`[84]
`[83]
`[87]
`[87]
`[45]
`[98]
`
`[47]
`[50]
`[99]
`
`[100]
`[100]
`
`Green- and blue-fluorescent protein
`fusions
`b-galactosidase and b-galactosidase
`fusion proteins
`Di-hydropholate reductase
`Endoglucanase D
`b-lactamase
`HtrA1 serine protease
`Interleukin-1 b
`Several a-helix-rich hyperthermophilic
`proteins
`TEM b-lactamase
`Lipase
`Human granulocyte-colony
`stimulating factor
`Human growth hormone
`Human interferon a 2b
`aFTIR, Fourier transformed infrared spectroscopy.
`bNMR, nuclear magnetic resonance.
`cCD, circular dychroism.
`
`www.sciencedirect.com
`
`Native-like secondary structure (FTIR)a
`Native-like secondary structure
`(FTIR; NMR; CD)b,c
`Native-like secondary structure (FTIR)
`Native-like secondary structure (FTIR)
`Native-like secondary structure (FTIR)
`
`Native-like secondary structure (FTIR)
`Native-like secondary structure (FTIR)
`
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`183
`
`reduce the average specific activity of the recombinant
`enzyme in the soluble cell
`fraction. Contrarily, an
`important part of the IB protein population must be
`properly folded and coexist with the background inter-
`molecular b-sheet organization [49] (Figure 1). Again, this
`might be indicative of conformational variability within
`IBs as a result of either native-like and b-enriched
`polypeptides, polypeptides trapped by b-enriched aggre-
`gation determinants (but keeping properly folded active
`site domains), or a combination of both. Although the
`specific activity of IB enzymes relative to their soluble
`versions is highly variable when comparing different
`proteins (Table 2), IBs formed by enzymes seem to be
`immediately useful in bioprocesses; they can skip any
`refolding step because their porous nature would permit
`substrate processing by the active enzyme molecules [84].
`Importantly, the availability of IbpAB and its occurrence
`in enzyme IBs significantly enhances their biological
`activities [87]. This observation confirms that these
`small heat-shock proteins, believed to preserve the
`folding-competent state of target proteins [88] and keep
`them suitable for refolding [67,72], are also efficient at
`preserving their native structure within aggregates.
`
`Conclusions and future prospects
`Rather than being ‘scrambled eggs’, bacterial inclusion
`bodies are dynamic and conformationally diverse struc-
`tures, formed by a sequence-selective aggregation process
`that is probably driven by certain ‘hot spots’ within the
`protein sequence. Furthermore, neither are they the dead-
`end of deficient folding processes but rather the transient
`reservoirs of aggregated polypeptides that are still under
`the quality control surveillance of cell chaperones and
`proteases. Recent insights into IB structure reveal that
`native or native-like proteins, or protein domains, coexist
`with b-sheet-rich intermolecular assemblies that share
`functional and architectural features with amyloid aggre-
`gates. In addition, the biological activity of enzymes and
`fluorescent proteins forming IBs is not dramatically lower
`than their soluble counterparts. Deeper exploration of this
`fact will
`open intriguing
`possibilities
`for
`the
`biotechnological industry.
`
`Acknowledgements
`AV acknowledges the support for research on protein aggregation through
`grants BIO2004–00700 (MEC; http://www.mec.es/) and 2005SGR-00956
`(AGAUR; http://agaur.gencat.net/). SV is recipient of a ‘Ramo´n y Cajal’
`contract awarded by the MCYT-Spain and co-financed by the Universitat
`Auto`noma de Barcelona (UAB; http://www.uab.es/), and founded by
`PNL2004–40 (UAB) and 2005SGR-00037(AGAUR).
`
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