BMC Biochemistry
`
`BioMed Central
`
`Open Access
`Research article
`Characterization of the aggregates formed during recombinant
`protein expression in bacteria
`Andrea Schrödel and Ario de Marco*
`
`Address: EMBL, Protein Expression Core Facility, Meyerhofstr. 1, D-69117, Heidelberg – Germany
`
`Email: Andrea Schrödel - andrea.schroedel@gmx.de; Ario de Marco* - ario.demarco@embl.de
`* Corresponding author
`
`Published: 31 May 2005
`
`BMC Biochemistry 2005, 6:10
`
`doi:10.1186/1471-2091-6-10
`
`This article is available from: http://www.biomedcentral.com/1471-2091/6/10
`
`Received: 10 February 2005
`Accepted: 31 May 2005
`
`© 2005 Schrödel and de Marco; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: The first aim of the work was to analyze in detail the complexity of the aggregates
`formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded
`in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and
`biophysical features, providing a novel approach for studying recombinant protein aggregates.
`Results: The total lysate separated into 4 different fractions whereas only the one with the lowest
`density was detected when the supernatant recovered after ultracentrifugation was loaded onto
`the sucrose gradient. The three further aggregate sub-classes were otherwise indistinctly
`precipitated in the pellet. The distribution of the recombinant protein among the four subclasses
`was strongly dependent on the DnaK availability, with larger aggregates formed in Dnak- mutants.
`The aggregation state of the GFP-GST recovered from each of the four fractions was further
`characterized by examining three independent biochemical parameters. All of them showed an
`increased complexity of the recombinant protein aggregates starting from the top of the sucrose
`gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also
`confirmed by electron microscopy analysis of the macro-structure formed by the different
`aggregates. Large fibrils were rapidly assembled when the recombinant protein was incubated in
`the presence of cellular extracts, but the GFP-GST fusion purified soon after lysis failed to undergo
`amyloidation, indicating that other cell components probably participate in the active formation of
`large aggregates. Finally, we showed that aggregates of lower complexity are more efficiently
`disaggregated by a combination of molecular chaperones.
`Conclusion: An additional analytical tool is now available to investigate the aggregation process
`and separate subclasses by their mass. It was possible to demonstrate the complexity of the
`aggregation pattern of a recombinant protein expressed in bacteria and to characterize
`biochemically the different aggregate subclasses. Furthermore, we have obtained evidence that the
`cellular environment plays a role in the development of the aggregates and the problem of the
`artifact generation of aggregates has been discussed using in vitro models. Finally, the possibility of
`separating aggregate fractions with different complexities offers new options for biotechnological
`strategies aimed at improving the yield of folded and active recombinant proteins.
`
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`
`Background
`The concept of protein aggregation suggests a non-physi-
`ological process resulting in the formation of large struc-
`tures, often chaotic, and in which the proteins have lost
`their original function/activity. Nevertheless, the collapse
`of the native conformation can also produce very regular
`structures, as in the case of amyloid fibrils [1]. Such a
`process can originate from sensitive protein intermediates
`during folding as well as from partially denatured proteins
`that lost their native conformation as a consequence of
`stress conditions.
`
`Cells possess a sophisticated quality control system to pre-
`vent the accumulation of protein aggregates. Molecular
`chaperones are engaged to promote the correct (re)-fold-
`ing of misfolded molecules that otherwise undergo pro-
`tease degradation. Misfolded proteins escaping the quality
`control may form aggregates that can be trapped in precip-
`itates (aggresome in eukaryotic cells, inclusion bodies in
`bacteria) to limit their interference with the cell physiol-
`ogy [2]. Inclusion bodies also have a storage function and
`parts of the trapped proteins are in a dynamic equilibrium
`with their soluble fraction [3]. Under pathological condi-
`tions aggregates develop into structures that hinder the
`cell functions, as in the case of neuron degenerative
`diseases.
`
`In bacteria the stress-dependent development of aggre-
`gates has been exploited to study the function of the chap-
`erone network. Aggregation has been reversed in vivo and
`the identification of the chaperone combinations neces-
`sary for the re-folding of the proteins from aggregates was
`performed using in vitro conditions [4-7]. Nevertheless,
`the biophysical features of the aggregates have never been
`investigated. Heat shock is the most studied stress factor
`but recombinant protein expression can also dramatically
`modify the cell balance. In fact, the exploitation of highly
`efficient polymerases increases the rate of protein synthe-
`sis so that as much as 50% of the totally accumulated pro-
`tein can be represented by the recombinant one and the
`cell folding machinery can become limiting. The optimi-
`zation of some growth parameters, like the use of low
`growth temperatures and non-saturating amounts of
`expression inducer as well as the over-expression of chap-
`erones by means of short heat shock, ethanol stress or
`recombinant co-expression [8,9], has often improved the
`yields of recombinant soluble proteins. Nevertheless, in
`most of the cases part or all of the recombinant protein
`expressed in bacteria is recovered as precipitates in the
`inclusion bodies.
`
`Both amorphous and organized inclusion bodies have
`been isolated [10]. Their composition varies from almost
`homogeneous to cases in which 50% of the material is
`represented by contaminants [11,12]. The structural het-
`
`erogeneity of the inclusion bodies has recently been
`shown [13,14] and it could be a consequence of the vari-
`able aggregation pattern to which a single protein can
`undergo under different conditions [15]. Proteins trapped
`in the inclusion bodies can be re-solubilised in vivo by
`impairing the de novo protein synthesis because the block
`of new protein production makes available larger
`amounts of chaperones and foldases for refolding precip-
`itated proteins [3]. The temporal separation between
`recombinant expression of chaperones and target proteins
`has also been successfully used to improve the yield of sol-
`uble recombinant proteins [8]. These results suggest a
`model for which soluble proteins are in a dynamic equi-
`librium with aggregates. In conclusion, modifications of
`the cell conditions can modulate the aggregation rate and
`the protein aggregation process can be reversed by condi-
`tions favorable for the folding machinery.
`
`This dynamic view for which proteins can pass from solu-
`ble to insoluble and back to soluble state suggests the
`presence of different degrees of aggregation complexity.
`Soluble aggregates of recombinant proteins have been
`described [16,17] and in a recent paper we have shown
`that the GFP-GST fusion protein expressed in bacteria
`forms aggregates with an estimated mass ranging from a
`few hundred kDa to more than 1000 kDa [18]. The sepa-
`ration of the aggregates using a blue native gel electro-
`phoresis followed by SDS-PAGE indicated an almost
`continuous distribution with few regions of concentrated
`accumulation. This kind of analysis allows for precise
`identification of aggregate patterns and comparison
`among different samples but is not suitable for the further
`characterization of the aggregates. Therefore, we present
`here an alternative protocol to separate sub-classes of
`aggregates using a sucrose step gradient and the results
`concerning the biophysical organization and biochemical
`specificities of such aggregates.
`
`Results and Discussion
`Separation of protein aggregate sub-classes by sucrose
`step gradient
`Preliminary experiments showed that the recombinant
`GFP-GST produced in bacteria grown at temperature
`higher than 30°C was mainly recovered in the pellet after
`ultracentrifugation of the lysates. Nevertheless, decreasing
`growth temperatures enabled the proportionally inversed
`recovery of the fusion protein in the supernatant. At 20°C
`roughly half of the total GFP-GST was in the supernatant
`(data not shown).
`
`Density gradients have been widely used to separate bio-
`logical material according to mass. We loaded cell frac-
`tions from bacteria induced to express the GFP-GST fusion
`recombinant protein on a sucrose step gradient to recover
`sub-classes of aggregates. The fluorescence of GFP-GST
`
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`Fraction 4
`
`Fraction 3
`
`Fraction 2
`
`Fraction 1
`
`B)
`
`DnaK
`
`ClpB
`
`IbpB
`
`GroEL
`
`sucrose fractions
`
`0%
`
`30%
`
`50%
`
`70%
`
`80%
`
`1 2 3 4
`
`DnaKoverexpression30°C
`DnaK-
`total lysate30°C
`
`mutant20°C
`
`total lysate20°C
`
`supernatant 20°C
`
`1
`
`2
`
`3
`
`4
`
`5
`
`A)
`
`Separation of recombinant GFP-GST fractions by a sucrose step gradientFigure 1
`
`Separation of recombinant GFP-GST fractions by a sucrose step gradient. A) Distribution of the recombinant protein using cell
`fractions recovered from different bacterial strains and from bacteria grown at different temperatures. Tube number 1 was
`loaded with the supernatant separated after lysate ultracentrifugation while total lysates were used for the other experiments.
`B) Dot-blot for the fractions separated by sucrose step gradient. Each fraction was tested with specific antibodies for the chap-
`erones DnaK, ClpB, IbpB and GroEL.
`
`simplified the identification of the sucrose concentrations
`which enabled the separation of the aggregates only at the
`interface between two different sucrose cushions. Finally,
`four fractions of GFP-GST were separated when loading a
`total lysate recovered from bacteria grown at 20°C onto a
`0%, 30%, 50%, 70%, 80% sucrose step gradient (Fig. 1A,
`tube number 2). SDS analysis confirmed that the recom-
`binant GFP-GST was the major protein in all the fractions,
`however, the co-migrated bacterial proteins were specific
`for a particular fraction (data not shown). We have
`already shown that aggregates of GFP-GST can trap other
`proteins [18] and that chaperones can strongly bind to
`aggregated recombinant proteins [19]. Dot blot analysis
`performed using antibodies against the major chaperones
`showed that DnaK and ClpB were concentrated mostly in
`
`the upper gradient fractions -in which the low-density
`material accumulated- while GroEL and IbpB co-migrated
`with the larger GFP-GST aggregates (Fig. 1B). These data
`are in agreement with previous reports that indicated a
`preferential binding of the different chaperones to aggre-
`gates with different degree of complexity [6,7].
`
`The recombinant protein from the four fractions was puri-
`fied by metal affinity chromatography and both fluores-
`cence and SDS-PAGE analysis indicated that the entire
`recombinant protein was bound and specifically eluted
`(data not shown). Protein amount determined by Brad-
`ford indicated that, on average, 39% of the total GFP-GST
`accumulated in the fraction 1, 14%, 22% and 25% in the
`other three, respectively, from the top to the bottom.
`
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`Table 1: Biophysical characterization of the different aggregate fractions separated by sucrose gradient. The 4 fractions were analysed
`for their aggregation index, their elution profile using size exclusion chromatography (SEC) and calculating the ratio between
`aggregated and monodispersed protein, and their binding to the dye ThioflavinT, indicative of amyloid formation. The results refer to
`one experiment representative of three repetitions.
`
`Aggregation index Abs 280/340 nm SEC index monodispersed/ aggregated protein
`
`ThioflavinT Abs 482 nm
`
`Fraction 1
`Fraction 2
`Fraction 3
`Fraction 4
`
`0.38
`2.83
`3.95
`5.96
`
`1.8
`0.5
`0.4
`0.25
`
`4.8
`8.8
`9.6
`13.4
`
`After ultracentrifugation of the lysate, the supernatant was
`loaded onto the sucrose gradient and the GFP-GST
`migrated exclusively to the interface between 0% and 30%
`sucrose (Fig. 1A, tube number 1). We knew from the pre-
`liminary experiments that bacteria grown at 30°C pro-
`duced only insoluble GFP-GST. The fusion protein
`present in the total lysate from such bacteria was distrib-
`uted almost exclusively in the fractions 3 and 4 and the
`fluorescence was almost undetectable (Fig. 1A, tube
`number 3).
`
`The role of chaperones in limiting the protein aggregation
`has been widely demonstrated and DnaK has a key role in
`the chaperone network [4-7]. The sucrose step gradient
`demonstrated what kind of aggregate pattern modifica-
`tions occur when the DnaK concentrations vary. No GFP-
`GST was recovered anymore in the upper fraction when
`DnaK- mutant bacteria were grown at 20°C and non-fluo-
`rescent aggregates largely accumulated in the lower frac-
`tions and even on the bottom of the tube (Fig. 1A, tube
`number 4). In contrast, both soluble GFP-GST and
`stronger fluorescence were detected after separation of a
`lysate from bacteria over-expressing DnaK grown at 30°C
`(Fig. 1A, tube number 5), suggesting that DnaK can
`improve the GFP-GST stability.
`
`This first set of experiments showed the complexity of the
`aggregation pattern. In fact, the previously non-character-
`ized insoluble fraction recovered in the pellet was distrib-
`uted in three classes according to mass and it was possible
`to separate soluble and insoluble recombinant protein by
`means of a sucrose gradient. Noteworthy is also the fact
`that fluorescence can be found in all the four fractions
`(Fig. 1A), indicating that even in the insoluble aggregates
`of a larger mass at least part of the trapped recombinant
`protein conserved a native-like structure. This is in agree-
`ment with the report that part of the protein present in the
`inclusion bodies conserves its secondary structure [20].
`Aggregate sub-classes with different complexity and pro-
`tease resistance have previously been identified in inclu-
`sion bodies and also in that case a protein fraction was
`still active [13,14,21]. In this study, the structural hetereo-
`
`genity of the proteins trapped in the aggregates is con-
`firmed by our data.
`
`Biophysical characterization of the GFP-GST fractions
`separated by the sucrose gradient
`The separation of the recombinant GFP-GST on the
`sucrose gradient is an indication of a mass difference
`among the aggregates and we wished to confirm these
`data by size exclusion chromatography (SEC). First, the
`GFP-GST proteins affinity purified from the four sucrose
`gradient fractions were dialysed and analysed in the fluo-
`rimeter according to the method proposed by Nominé et
`al. [22], namely the absorbance at 280 and 340 nm was
`measured and the ratio calculated. This value (aggregation
`index) indicates the relative aggregation, is quickly deter-
`mined, and allows the comparison of different fractions
`of the same protein. Low values indicate a lower aggrega-
`tion state and our data show that there is a gradient of
`increasing aggregation from the top fraction to the bottom
`fractions (Table 1).
`
`The 4 GFP-GST fractions were also subjected to SEC and
`the ratio between the areas of the peaks corresponding to
`the monodispersed and the aggregated protein was calcu-
`lated (SEC index). Such an index confirmed an increasing
`state of aggregation from sucrose fraction 1 to 4 (Table 1).
`Surprisingly, the SEC experiments showed that both
`aggregated and functional forms of the fusion protein
`were present in both the three fractions corresponding to
`the insoluble GFP-GST and the (soluble) fraction 1. Solu-
`ble aggregates have been described before and are proba-
`bly common when fusion proteins are expressed [16,17].
`It was not possible to separate monodispersed GFP-GST
`from soluble aggregates by means of sucrose gradients of
`decreasing concentrations (data not shown).
`
`We finally tried to characterize the aggregates according to
`their specific structure. ThioflavinT (ThT) is a dye that
`preferentially binds to amyloid-like fibrils [23]. We meas-
`ured an increasing binding when aggregates of higher
`complexity were used (Table 1). In contrast, there was not
`significant binding of any aggregate to 8-anilino-1-
`
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`naphtalenesulfonic acid (ANSA) that has been used as a
`marker of the amorphous aggregates [24]. This suggests
`that the aggregates formed by GFP-GST probably have a
`regular structure involving β-sheets rather than being a
`chaotic complex held together by hydrophobic interac-
`tions. Instead, a micellar organization has been proposed
`for the soluble aggregates [17,22].
`
`Aggregate identification by electron microscopy
`In the case of the GFP-GST fractions we showed that the
`degree of amyloidation detected by ThT-binding progres-
`sively increased from fraction 1 to fraction 4 (Table 1).
`The capacity to form fibrils is sequence specific [25] and it
`seems a generic feature of polypeptide chains [26]. The
`development into fibrils is characterized by a log phase
`during which the aggregation seeds are formed followed
`by a period of rapid growth [27]. Once formed, the fibrils
`act as aggregation seeds, speeding up the process. There-
`fore, it could be expected that larger aggregate networks
`have the possibility to develop faster into structures of
`higher complexity. In order to test this hypothesis, the
`GFP-GST from the four sucrose gradient fractions was
`recovered immediately after centrifugation and mounted
`for electron microscopy analysis.
`
`Some aggregation seeds (20–40 nm in diameter) were vis-
`ible even when the GFP-GST from the upper fraction was
`used (Figure 2A, fraction 1). Sort of chains composed by
`globular elementary structures and measuring several
`hundreds of nm were observed when GFP-GST from the
`fraction 2 was exploited (Figure 2A) while protofilaments
`and higher ordered fibrils [28] longer than 1 µm (Figure
`2A) were visible when samples from fractions 3 and 4
`were used. Therefore, it was possible to demonstrate the
`relation between the biochemical indexes used to charac-
`terize the aggregation of GFP-GST and the macro-aggrega-
`tion complexity visible by electron microscopy.
`
`Fibrils are the end product of GFP-GST aggregation but
`the different classes of aggregates separated by sucrose gra-
`dient can be considered as dynamic intermediates that can
`either develop to larger structures or be reversed into
`lower-complexity aggregates [29]. Both the initial com-
`plexity and the incubation time of polypeptides prone to
`aggregation are crucial for the building of the aggregates.
`We wished to demonstrate the importance of these factors
`in a control experiment. GFP-GST was separated into frac-
`tions by sucrose gradient and the fractions 1 and 4 were
`mounted for electron microscopy only after 24 hours of
`incubation in the presence of the co-migrated cell compo-
`nents. Both samples raised similar large fibrils (Figure
`2B), indicating that the incubation period was sufficient
`for both, independent of their initial aggregation state, to
`reach the rapid growth phase that leads to the fibril
`formation.
`
`This experiment underlines once more the importance of
`the parameter time in studies dealing with aggregation
`and questions the meaning of some in vitro experiments.
`In fact, the fibril maturation outside the bacterial cell
`could have peculiar features. For instance, the lack of
`space-constrain or limitations in the disaggregation proc-
`esses could enable the formation of fibrils the length of
`which are difficultly compatible with the size of E. coli
`cells (Figure 2B). The experiments described in the two
`last paragraphs will show the impact of cell components
`in promoting aggregation and disaggregation.
`
`Finally, the presence of aggregation seeds smaller than 40
`nm in diameter shows that it is not possible to discrimi-
`nate between soluble and aggregated fractions by the use
`of simplified methods in high-throughput protocols as,
`for instance, the exploitation of a 0.65 µm pore size filter
`[30].
`
`Is the aggregation of GFP-GST actively supported?
`In the previous experiments we showed that even the
`moderately aggregated GFP-GST recovered from the upper
`fraction of the sucrose gradient could form fibrils if the
`sample was incubated with the cell fraction for at least 1
`day before it was prepared for the electron microscopy
`analysis. In a recent paper it was claimed that bacterial
`chaperones play an active role in the formation of the
`aggregates [31]. The possible participation of cell compo-
`nents in catalyzing the GFP-GST fibril formation was
`investigated in a control experiment. The process of aggre-
`gate maturation of the soluble recombinant protein in the
`presence of other cell components was limited to 1 hour
`performing the affinity purification of the GFP-GST
`immediately after lysis to avoid a seeding process during
`the 15 hour centrifugation of the cell components upon
`the sucrose gradient. The sample was incubated at room
`temperature for 4 weeks and the modifications of the sec-
`ondary structure were monitored by CD while corre-
`sponding samples were mounted for electron microscopy.
`No significant modification was observed in the first two
`weeks and a slight increase of the β-sheet content was
`measured only after 4 weeks (Figure 3). The use of differ-
`ent protein concentrations and the addition of sucrose to
`the proteins did not modify the pattern and no detectable
`aggregate was observed at the electron microscopy using
`the corresponding samples (data not shown).
`
`Therefore, these results strongly suggest that the co-pres-
`ence of other molecules is necessary to trigger the process
`of regular aggregation of the recombinant protein,
`probably by facilitating the formation of aggregation
`seeds. Chaperones can play a role in the aggresome forma-
`tion [32] and GroEL has been claimed to be actively
`involved in bacterial inclusion body formation [31]. Our
`data can only confirm that GroEL co-migrates with the
`
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`A)
`
`Fraction 1
`
`Fraction 2
`
`B)
`
`Fraction 1
`
`200nm
`
`Fraction 3
`
`Fraction 4
`
`Fraction 4
`
`Electron microscopy characterization of GFP-GST macro-aggregatesFigure 2
`
`Electron microscopy characterization of GFP-GST macro-aggregates. A) Samples recovered from the 4 aggregation fractions
`were mounted soon after the sucrose gradient separation and observed by electron microscopy. B) The samples for the elec-
`tron microscopy grids were from the fractions 1 and 4 recovered after sucrose gradient separation but incubated 24 hours
`with the co-migrated cell fraction before being mounted.
`
`aggregates of larger mass (Fig. 1B). Finally, we are looking
`for an analytical method to determine if the process of cell
`lysis is crucial for the development of the aggregates.
`
`Aggregate complexity and re-folding
`Both in vivo and in vitro experiments illustrated the co-
`operative action of chaperone networks in disaggregating
`misfolded proteins [4-7] but the features of the real aggre-
`gates that are the target of the chaperones in the cells have
`never been investigated. We used the aggregates from frac-
`tions 3 and 4 to test if they could be a substrate for chap-
`erone-dependent refolding and if the different structure
`complexity had a role on the refolding kinetic.
`
`An equimolar combination of DnaK, DnaJ, GrpE, and
`ClpB [6] quickly disaggregated the large precipitates (Fig-
`
`ure 4). Specifically, the complexity of the aggregates from
`fraction 3 was reduced in a faster and more efficient way.
`In fact, the aggregation index dropped by half in only 4
`min while it took 10 min in the case of the aggregates
`from fraction 4. Furthermore, there was a higher residual
`aggregation: the aggregation indexes measured were 1.2
`and 0.7 for the aggregates from fractions 4 and 3, respec-
`tively. In comparison, the GFP-GST from fraction 1 scored
`0.38 (Table 1). The addition of equimolar amounts of
`BSA to the aggregates in absence of chaperones had no
`disaggregation effect.
`
`The preferential disaggregation of subclasses of aggregates
`with lower complexity observed in vitro is reminiscent of
`previous works indicating that specific subclasses of the
`proteins trapped in the inclusion bodies are preferentially
`
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`CD spectra of purified GFP-GST
`
`Day 0
`
`-
`
`Day 10 -
`
`Day 15 -
`
`Day 27 -
`
`200
`
`205
`
`210
`
`215
`
`220
`
`225
`
`230
`
`235
`
`240
`
`245
`
`250
`
`Wavelength (nm)
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`-5
`
`-10
`
`-15
`
`-20
`195
`
`(degxcm2xdmol-1) x 104
`
`Ellipticity
`
`Circular dichroism spectra of GFP-GSTFigure 3
`
`Circular dichroism spectra of GFP-GST. Protein purified by metal affinity from the supernatant obtained after lysate ultracen-
`trifugation was directly analysed (day 0) or incubated at room temperature before the collection of further spectra the days 10,
`15, and 27.
`
`refolded under physiological conditions [3,13] and that
`the reversibility is increasingly difficult and dependent on
`the size of the aggregates [29]. The limit of this experiment
`is that it is difficult to scale up and the small amount of
`the protein used was insufficient for undertaking further
`biophysical analysis. The aggregation index gives only rel-
`ative values and, therefore, we can state that the degree of
`aggregation decreased but cannot conclude that the disag-
`gregated protein was also correctly folded. Nevertheless,
`the results suggest that it would be of biotechnological
`interest to separate the aggregate subclasses and use the
`lower complexity aggregates in refolding protocols.
`
`Conclusion
`There is increasing evidence that aggregates are heteroge-
`neous in size and complexity [2,12-16,26]. The aggre-
`
`somes are actively built in eukaryotic cells and the
`physiological meaning of the process would be the pack-
`ing of disorganized aggregates that could interfere with
`the normal cell functions by non-specifically binding to
`other cell components [33,34]. The possibility to recover
`functional proteins from the insoluble aggregates [3]
`would indicate that at least in bacteria they can function
`as a reserve in dynamic equilibrium with soluble
`fractions.
`
`The expression of recombinant proteins is a stress factor
`because they compete for energy and substrates with
`native expression and can interfere with the normal
`metabolism by forming aggregates, both in prokaryotic
`and eukaryotic cells [2,34]. The possibility to store the
`excess of misfolded recombinant protein could be a way
`
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`accepted and our separation protocol turned out to be a
`useful tool for characterizing the aggregates. Furthermore,
`such an aggregation process shares many features with the
`maturation of pathological amyloids in eukaryotic cells
`and, therefore, the bacterial system -experimentally easy
`to modify- would be considered as a model to integrate
`the results obtained using in vitro systems and to study the
`impact of chemical and biophysical parameters on the
`aggregation development. We simplified the work by
`using a fluorescent construct but any protein for which
`antibodies are available could be used for following the
`aggregation development.
`
`Methods
`Cell culture and protein preparation
`A fusion construct His-GST-GFP cloned in a Gateway des-
`tination vector (Invitrogen, kindly provided by D.
`Waugh) was transformed and expressed in the following
`bacterial strains: BL 21 (DE3), BL 21 (DE3) RIL codon
`plus, GK2 (dnak-), BL 21 (DL3) co-expressing the chaper-
`one combinations GroELS and GroELS/DnaK/DnaJ/
`GrpE/ClpB, respectively (kindly provided by B. Bukau).
`Bacteria were grown at 37°C until the OD600 reached 0.4,
`then the cultures were adapted to different temperatures
`(20°C, 25°C, 30°C, 37°C), induced at an OD600 of 0.6
`with 0.1 mM IPTG and grown for further 20 h. The bacte-
`ria were pelleted by centrifugation (6000 g × 15 min),
`washed in 10 mL of PBS and finally stored at -20°C.
`
`The pellet was resuspended in 10 mL of lysis-buffer (50
`mM potassium phosphate buffer, pH 7.8, 0.5 M NaCl, 5
`mM MgCl2, 1 mg/mL lysozyme, 10 µg/mL DNase), soni-
`cated in a water bath (Branson 200) for 5 min and the
`lysate was incubated for 30 min on a shaker at room tem-
`perature. The supernatant was recovered after ultracentrif-
`ugation (35 min at 150000 × g).
`
`Fractions from sucrose gradients were recovered using a
`bent Pasteur pipette and affinity purified using a HiTrap
`chelating affinity column (Amersham Biosciences) pre-
`equilibrated with 20 mM Tris HCl, pH 7.8, 500 mM NaCl,
`15 mM imidazole. The His-tagged recombinant protein
`was eluted in 20 mM Tris, pH 7.8, 125 mM NaCl, and 250
`mM imidazole. Protein quantification was based on the
`absorbance at 280 nm.
`
`Sucrose gradients and gel filtration
`Total cell lysates or supernatants from ultracentrifugation
`of total cell lysates (1 mL) were loaded onto 14 × 95 mm
`Ultra-Clear centrifuge tubes (Beckman) prepared with a
`step gradient formed by four layers of 20 mM TrisHCl
`buffer, pH 8, containing 80%, 70%, 50%, 30%, and 0%
`sucrose, respectively. The tubes were centrifuged 15 hours
`at 180,000 × g at 4°C using a SW40Ti rotor and a L-70
`Beckman ultracentrifuge. The protein fractions were
`
`Page 8 of 11
`(page number not for citation purposes)
`
`agg3
`
`agg4
`
`0123456789
`
`Aggregation index
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`min
`
`Chaperone-dependent in vitro disaggregationFigure 4
`
`Chaperone-dependent in vitro disaggregation. Purified GFP-
`GST aggregates recovered from the fractions 3 and 4 of the
`sucrose gradient were incubated in the presence of an equi-
`molar mixture of DnaK, DnaJ, GrpE, and ClpB in the pres-
`ence of a system constantly providing ATP. The aggregation
`index was repeatedly measured during a 45 min incubation.
`
`to get rid of dangerous aggregating material when mis-
`folded proteins escaped the quality control of chaperones
`and proteases [2]. The cellular mechanisms that favor the
`generation of amyloids (Figure 2) might also be useful in
`preventing amorphous aggregates in non-specifically trap-
`ping native proteins [18]. The aggregate organization
`would consider an aggregate mash that grow from small
`entities towards larger insoluble structures [34] composed
`by a core of protease-resistant fibrils [13,14], homologous
`proteins at different levels of misfolding and some heter-
`ologous and non-specifically trapped proteins [18] (Fig-
`ure 5B).
`
`In this paper we present data supporting the idea of a pro-
`gressive maturation of recombinant GFP-GST aggregates
`into amyloid fibrils. Furthermore, it seems that the proc-
`ess is facilitated by some other cell components since the
`fibril maturation was extremely slower when the recom-
`binant protein was separated from the other cell compo-
`nents soon after the lysis (Fig. 3). For instance, GroEL has
`been reported having an active role in inclusion body for-
`mation [31] and specifically co-migrate with the larger
`aggregates could (Fig. 1B). Conversely, the combination
`of DnaK, DnaJ, GrpE and ClpB could disaggregate large
`insoluble structures (Figures 4 and 5A).
`
`It seems that the aggregation process of recombinant pro-
`teins is extremely more complicated than normally
`
`GNE 2008
`Page 8
`
`

`
`BMC Biochemistry 2005, 6:10
`
`http://www.biomedcentral.com/1471-2091/6/10
`
`A)
`
`B)
`
`Folded
`
`Aggregate model
`
`Folded GFP-GST
`GFP-GST fibrils
`
`Unfolded GFP-GST
`
`chaperones
`
`I
`
`II
`
`III
`
`IV
`
`Aggregation classes
`
`Soluble
`
`Insoluble
`
`Inclusion bodies
`
`Other proteins
`
`Schematic representation of the aggregationFigure 5
`
`Schematic representation of the aggregation. A) Dynamic of the aggregation. GFP-GST aggregates progressively form both sol-
`uble and insoluble aggregates. Chaperone activity can reverse the process of aggregation in