BIVIC Biochemistry
`
`magenta.
`
`Research article
`
`Characterization of the aggregates formed during recombinant
`protein expression in bacteria
`Andrea Schrodel and Ario de Marco*
`
`Address: EMBL Protein Expression Core Farilily. Mcycrhofslr. I, 0—69] I7. Heidelberg — Cennany
`
`Email: Andrea Srlirt‘idal — andreasrhmedal@gmxxle; Ario de Marrof — ario.tleinarm@cmbl.de
`* Corresponding author
`
`Published: 3 | May 2005
`doi: |0.| |86i|4?| 409 l—é—l u
`BMC Biochemisz 2005. a: lo
`This article is available from: http:ffwww.biomedcentralcomi’ l47| -209 ”6:" IO
`© 2005 Schrodel and de Marco; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution license (W3).
`which permits unrestricted use, distribution, and reproduction in any medium. provided the original work is properly cited.
`
`Received: IO Februaryr 2005
`AccePte‘l: 3' "1" 2005
`
`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 tap of the sucrose
`gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also
`confirmed by electron microsc0py 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 deveIOpment 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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`BMC Biochemistry 2005, 6:10
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`http://www.biomedcentral.com/1471-2091/6/1O
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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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`BMC Biochemistry 2005, 6:10
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`A)
`
`
`
`DnaKoverexpression30°C
`
`
`
`sucrosefractions
`
`Fraction1 Fraction2 Fraction3 Fraction4
`
`
`
`
`
`DnaK'mutant20°C
`
`
`
`
`
`
`
`totallysate20°C
`
`
`
`
`
`“’totallysate30°C
`
`0O
`
`ON4
`
`.-
`
`C(
`
`B4.-
`{B
`CL
`
`oD
`
`.
`
`3(
`
`0
`
`Figure I
`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 I 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, CIpB. lpr and GroEL.
`
`simplified the identification ofthe sucrose concentrations
`which enabled the separation of the aggregates only at the
`interface between two different sucrose cushions. Finally,
`four fractions of GFP—CST 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 continued 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 CFP—GST can trap other
`proteins [18] and that chaperones can stroneg bind to
`aggregated recombinant proteins [19]. [)ot 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 [pr co—migrated
`with the larger EFF—CST aggregates (Fig. lli). 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 SPF-(Sr
`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 I: 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.
`
`ThioflavinT Abs 482 nm
`SEC index monodispersed/ aggregated protein
`Aggregation index Abs 280/340 nm
`
`
`0.38
`Fraction |
`4.8
`| .8
`8.8
`0.5
`2.83
`Fraction 2
`3.95
`Fraction 3
`9.6
`0.4
`| 3.4
`0.25
`5.96
`Fraction 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, 14,2 1]. 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 B—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 um (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 mm pore size filter
`[30].
`
`Is the aggregation of GFP-GS T 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.
`N0 significant modification was observed in the first two
`weeks and a slight increase of the B—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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`Fraction 2
`
`Fraction 1
`
`
`
`Fraction 4
`
`Fraction 4
`
`
`
`Figure 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 l and 4 recovered after sucrose gradient separation but incubated 24 hours
`with the co-migrated cell fraction before being mounted.
`
`aggregates oflarger mass (Fig. 1B). Finally, we are looking
`for an analytical method to determine ifthe process ofcell
`lysis is crucial for the development ofthe 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, Dna], 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—CSI‘ fIOm fraction ] 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 compledty observed in vitro is reminiscent of
`previous works indicating that specific subdasses of the
`proteins trapped in the inclusion bodies are preferentially
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`Ellipticity
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` (degxcmzxdmol'l)x104
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`BMC Biochemistry 2005, 6:10
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`CD spectra of purified GFP-GST
`
`
`
`
`DayO -
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`Day10 -
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`Day15 -
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`Day 27 -
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`-20
`195
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`200
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`205
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`210
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`215
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`220
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`225
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`230
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`235
`
`240
`
`245
`
`250
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`Wavelength (nm)
`
`
`Figure 3
`Circular dich roism 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 I0,
`l5. and 27.
`
`refolded under physiological conditions [3,13] and that
`the reversibility is increasingly difficult and dependent on
`the size ofthe aggregates [29]. The limit ofthis 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 subdasses and use the
`lower complexity aggregates in refolding protocols.
`
`Conclusion
`
`There is increasing evidence that aggregates are heteroge—
`neous in size and complexity [112—1626]. 'Ihe aggre—
`
`in eukaryotic cells and the
`somes are actively built
`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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`index
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`
`
` Aggregation
`
`
`
`min
`
`Figure 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, Dna], 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
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`BMC Biochemistry 2005, 6:10
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`http://www.biomedcentral.com/1471-2091/6/10
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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/Dnai/
`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 x 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 MgC12, 1 mg/mL lysozyme, 10 ug/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 x 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 x 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 x g at 4°C using a SW40Ti rotor and a L—70
`Beckman ultracentrifuge. The protein fractions were
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`Aggregate model
`
`Folded GFP-GST
`
`GFP-GST fibrils
`
`Other protein ~
`
`Figure 5
`Schematic representation of the aggregation. A) Dynamic of the aggregation. GFP-GST aggregates progressively form bod'l sol-
`uble and insoluble aggregates. Chaperone activity can reverse the process of aggregation in a way that is inversely pr0por'tional
`to the degree of complexity reached by the aggregates and could also play a role in the aggregate maturation towards more
`structured complexes. B) Aggregate model. The aggregation of GFP-GST probably starts widi misfolded single proteins that
`collapse into prewfibrillar structures. These catalyze the aggregation of new molecules to form larger amyloid fibrils. In the ini-
`tial phases, the co-presence of molecules with diFferent degree of misfolding and amyloidation seems apparent. Pre—fibrils could
`form the