b.tech. Biotechnology Notes (6)

8/6/2019 b.tech. Biotechnology Notes (6)

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ASSIGNMENTON

THECASE

STUDY OF

SEPERATIONOF

THEAGGREGATES

FORMEDDURING

RECOMBINANTPROTEIN

EXPRESSION

SUBMITTED BY:-MUDIT MISRAB.TECH(B.T.)SECTION-A



ROLL No.-33

SEPERATION OF THE AGGREGATES FORMED

DURING RECOMBINANT PROTEIN EXPRESSIONin bacteria

BackgroundThe first aim of the work was to analyze in detail thecomplexity of the aggregates formed uponoverexpression of recombinant proteins in E. coli. Asucrose step gradient succeeded in separatingaggregate subclasses of a GFP-GST fusion protein

with specific biochemical and biophysical features,providing a novel approach for studying recombinantprotein aggregates.

ResultsThe total lysate separated into 4 different fractionswhereas only the one with the lowest density wasdetected when the supernatant recovered afterultracentrifugation was loaded onto the sucrosegradient. The three further aggregate sub-classeswere otherwise indistinctly precipitated in the pellet.The distribution of the recombinant protein amongthe four subclasses was strongly dependent on theDnaK availability, with larger aggregates formed inDna k- mutants. The aggregation state of the GFP-GST recovered from each of the four fractions wasfurther characterized by examining threeindependent biochemical parameters. All of themshowed an increased complexity of the recombinant

protein aggregates starting from the top of thesucrose gradient (lower mass aggregates) to thebottom (larger mass aggregates). These results werealso confirmed by electron microscopy analysis of themacro-structure formed by the different aggregates.Large fibrils were rapidly assembled when therecombinant protein was incubated in the presence of

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cellular extracts, but the GFP-GST fusion purifiedsoon after lysis failed to undergo amyloidation,indicating that other cell components probablyparticipate in the active formation of large

aggregates. Finally, we showed that aggregates of lower complexity are more efficiently disaggregatedby a combination of molecular chaperones.

ConclusionAn additional analytical tool is now available toinvestigate the aggregation process and separate

subclasses by their mass. It was possible todemonstrate the complexity of the aggregationpattern of a recombinant protein expressed inbacteria and to characterize biochemically thedifferent aggregate subclasses. Furthermore, wehave obtained evidence that the cellular environmentplays a role in the development of the aggregates andthe problem of the artifact generation of aggregateshas been discussed using in vitro models. Finally, the

possibility of separating aggregate fractions withdifferent complexities offers new options forbiotechnological strategies aimed at improving theyield of folded and active recombinant proteins.

BackgroundThe concept of protein aggregation suggests a non-physiological process resulting in the formation of large structures, often chaotic, and in which the

proteins have lost their original function/activity.Nevertheless, the collapse of the native conformationcan also produce very regular structures, as in thecase of amyloid fibrils. Such a process can originatefrom sensitive protein intermediates during folding aswell as from partially denatured proteins that lost

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stress or recombinant co-expression, has oftenimproved the yields of recombinant soluble proteins.Nevertheless, in most of the cases part or all of therecombinant protein expressed in bacteria is

recovered as precipitates in the inclusion bodies.

Both amorphous and organized inclusion bodies havebeen isolated . Their composition varies from almosthomogeneous to cases in which 50% of the material isrepresented by contaminants. The structuralheterogeneity of the inclusion bodies has recentlybeen shown and it could be a consequence of thevariable aggregation pattern to which a single proteincan undergo under different conditions. Proteins

trapped in the inclusion bodies can be re-solubilisedin vivo by impairing the de novo protein synthesisbecause the block of new protein production makesavailable larger amounts of chaperones and foldasesfor refolding precipitated proteins . The temporalseparation between recombinant expression of chaperones and target proteins has also beensuccessfully used to improve the yield of solublerecombinant proteins. These results suggest a model

for which soluble proteins are in a dynamicequilibrium with aggregates. In conclusion,modifications of the cell conditions can modulate theaggregation rate and the protein aggregation processcan be reversed by conditions favorable for thefolding machinery.

This dynamic view for which proteins can pass fromsoluble to insoluble and back to soluble statesuggests the presence of different degrees of aggregation complexity. Soluble aggregates of recombinant proteins have been described and in arecent paper we have shown that the GFP-GST fusionprotein expressed in bacteria forms aggregates withan estimated mass ranging from a few hundred kDato more than 1000 kDa . The separation of the

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aggregates using a blue native gel electrophoresisfollowed by SDS-PAGE indicated an almost continuousdistribution with few regions of concentratedaccumulation. This kind of analysis allows for precise

identification of aggregate patterns and comparisonamong different samples but is not suitable for thefurther characterization of the aggregates. Therefore,we present here an alternative protocol to separatesub-classes of aggregates using a sucrose stepgradient and the results concerning the biophysicalorganization and biochemical specificities of suchaggregates.

Methods

Cell culture and protein preparation

A fusion construct His-GST-GFP cloned in a Gatewaydestination vector (Invitrogen, kindly provided by D.Waugh) was transformed and expressed in thefollowing bacterial strains: BL 21 (DE3), BL 21 (DE3)RIL codon plus, GK2 (dnak-), BL 21 (DL3) co-expressing the chaperone combinations GroELS and

GroELS/DnaK/DnaJ/GrpE/ClpB, respectively (kindlyprovided by B. Bukau). Bacteria were grown at 37°Cuntil the OD600 reached 0.4, then the cultures wereadapted to different temperatures (20°C, 25°C, 30°C,37°C), induced at an OD600 of 0.6 with 0.1 mM IPTGand grown for further 20 h. The bacteria werepelleted by centrifugation (6000 g × 15 min), washedin 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 MNaCl, 5 mM MgCl2, 1 mg/mL lysozyme, 10 μg/mLDNase), sonicated in a water bath (Branson 200) for 5min and the lysate was incubated for 30 min on ashaker at room temperature. The supernatant was

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recovered after ultracentrifugation (35 min at 150000× g).

Fractions from sucrose gradients were recoveredusing a bent Pasteur pipette and affinity purifiedusing a HiTrap chelating affinity column (AmershamBiosciences) pre-equilibrated with 20 mM Tris HCl, pH7.8, 500 mM NaCl, 15 mM imidazole. The His-taggedrecombinant protein was eluted in 20 mM Tris, pH 7.8,125 mM NaCl, and 250 mM imidazole. Proteinquantification was based on the absorbance at 280nm.

Sucrose gradients and gel filtration

Total cell lysates or supernatants fromultracentrifugation of total cell lysates (1 mL) wereloaded onto 14 × 95 mm Ultra-Clear centrifuge tubes(Beckman) prepared with a step gradient formed byfour layers of 20 mM TrisHCl buffer, pH 8, containing80%, 70%, 50%, 30%, and 0% sucrose, respectively.The tubes were centrifuged 15 hours at 180,000 × gat 4°C using a SW40Ti rotor and a L-70 Beckmanultracentrifuge. The protein fractions were recovered

from the interfaces between two sucrose layers,affinity purified as described above and used forfurther analysis. The samples for gel filtration wereconcentrated and the buffer replaced with 50 mMTrisHCl, pH8.0, 150 mM NaCl using a Vivaporeconcentrator (Vivascience) and then separated by gelfiltration using a Superose 12 HR 10/30 column(Amersham).

Bioanalytical assays

The aggregation rate of the proteins was analysedaccording to Nominé et al. [22] using an AB2Luminescence Spectrometer (Aminco Bowman Series2) equipped with SLM 4 software. The excitation was

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induced at 280 nm and the emission scan wasrecovered between 260 and 400 nm.

Amyloid aggregates were estimated according to

their binding to the specific dye thioflavin-T (ThT), asdescribed by LeVine [23], and protein surfacehydrophobicity was determined using the fluorescentprobe 8-anilino-1-naphtalenesulfonic acid (ANSA)[24].

Circular dichroism (CD) spectra were recordedbetween 250 and 190 nm using suprasil precisioncells (Hellma) and a Jasco J-710 instrument.

Western and dot blotting

Western blots were performed as previouslydescribed using anti-GST primary antibodies. For dotblotting the proteins were transferred onto a PVDFmembrane using a Bio-Rad Criterion blotter. Theprimary rabbit antibodies were a gift from Dr. Bukauand were purified from sera using Protein GPlus/Protein A Agarose (Oncogene) to minimize thebackground. Peroxidase-conjugated secondaryantibodies for chemioluminescent detection werepurchased from Dianova and the detection performedusing the SuperSignal® West Femto MaximumSensitivity Substrate (Pierce), following the supplier'sinstructions. Blots were used repeatedly byeffectively removing the antigen-antibody interactionusing the Western Blot Recycling it (Alpha DiagnosticInt.).

Sample preparation for electron microscopy

Protein samples were purified by affinitychromatography and equal amounts fixed by usingthe "single-droplet" parafilm protocol. 5 μL of eachprotein sample were pipetted on a grid (AgarScientific) and incubated 1 min at room temperature.

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Excess fluid was removed using filter paper, theunbound protein was washed and the grids wereplaced on a 50 μL drop of 1% uranyl acetate with thesection side downwards. Finally, the grids were dried,

placed in the grid-chamber and stored in desiccatorsbefore the samples were observed with a CM120BioTwin electron microscope (Philips).

In vitro re-folding assay

The conditions for the chaperone-dependentdisaggregation of GST-GFP in vitro were chosenaccording to Mogk et al. [35] and the process wasmonitored using the fluorimetric assay described

above [22]. 1 μM of aggregated protein wasresuspended in 50 mM Tris HCl, pH 7.5, 20 mM MgCl2,150 mM KCl, 2 mM DTT, in the presence of 1 μM ClpB,1 μM DnaK, 0.2 μM DnaJ, 0.1 μM GrpE, 3 mMphosphoenolpyruvate, and 20 ng/mL of pyruvatekinase. The reaction was started by the addition of 2mM NaATP.

Results and Discussion

Separation of protein aggregate sub-classes by sucrose stepgradient

Preliminary experiments showed that therecombinant GFP-GST produced in bacteria grown attemperature higher than 30°C was mainly recoveredin the pellet after ultracentrifugation of the lysates.Nevertheless, decreasing growth temperaturesenabled the proportionally inversed recovery of thefusion protein in the supernatant. At 20°C roughlyhalf of the total GFP-GST was in the supernatant(data not shown).

Density gradients have been widely used to separatebiological material according to mass. We loaded cellfractions from bacteria induced to express the GFP-GST fusion recombinant protein on a sucrose step

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gradient to recover sub-classes of aggregates. Thefluorescence of GFP-GST simplified the identificationof the sucrose concentrations which enabled theseparation of the aggregates only at the interface

between two different sucrose cushions. Finally, fourfractions of GFP-GST were separated when loading atotal lysate recovered from bacteria grown at 20°Conto a 0%, 30%, 50%, 70%, 80% sucrose step gradient(Figure tube number 2).

SDS analysis confirmed that the recombinant GFP-

GST was the major protein in all the fractions,however, the co-migrated bacterial proteins werespecific for a particular fraction (data not shown). Wehave already shown that aggregates of GFP-GST cantrap other proteins and that chaperones can stronglybind to aggregated recombinant proteins. Dot blotanalysis performed using antibodies against themajor chaperones showed that DnaK and ClpB wereconcentrated mostly in the upper gradient fractions

-in which the low-density material accumulated- whileGroEL and IbpB co-migrated with the larger GFP-GSTaggregates (Fig. 1B). These data are in agreementwith previous reports that indicated a preferentialbinding of the different chaperones to aggregateswith different degree of complexity.

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The recombinant protein from the four fractions waspurified by metal affinity chromatography and bothfluorescence and SDS-PAGE analysis indicated thatthe entire recombinant protein was bound and

specifically eluted (data not shown). Protein amountdetermined by Bradford indicated that, on average,39% of the total GFP-GST accumulated in the fraction1, 14%, 22% and 25% in the other three, respectively,from the top to the bottom.

After ultracentrifugation of the lysate, thesupernatant was loaded onto the sucrose gradientand the GFP-GST migrated exclusively to the interfacebetween 0% and 30% sucrose (Fig. 1A, tube number

1). We knew from the preliminary experiments thatbacteria grown at 30°C produced only insoluble GFP-GST. The fusion protein present in the total lysatefrom such bacteria was distributed almost exclusivelyin the fractions 3 and 4 and the fluorescence wasalmost undetectable (Fig. 1A, tube number 3).

The role of chaperones in limiting the proteinaggregation has been widely demonstrated and DnaK

has a key role in the chaperone network . The sucrosestep gradient demonstrated what kind of aggregatepattern modifications occur when the DnaK concentrations vary. No GFP-GST was recoveredanymore in the upper fraction when DnaK- mutantbacteria were grown at 20°C and non-fluorescentaggregates largely accumulated in the lower fractionsand even on the bottom of the tube (Fig. 1A, tubenumber 4). In contrast, both soluble GFP-GST andstronger fluorescence were detected after separationof a lysate from bacteria over-expressing DnaK grownat 30°C (Fig. 1A, tube number 5), suggesting thatDnaK can improve the GFP-GST stability.

This first set of experiments showed the complexityof the aggregation pattern. In fact, the previously

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non-characterized insoluble fraction recovered in thepellet was distributed in three classes according tomass and it was possible to separate soluble andinsoluble recombinant protein by means of a sucrose

gradient. Noteworthy is also the fact thatfluorescence can be found in all the four fractions(Fig. 1A), indicating that even in the insolubleaggregates of a larger mass at least part of thetrapped recombinant protein conserved a native-likestructure. This is in agreement with the report thatpart of the protein present in the inclusion bodiesconserves its secondary structure. Aggregate sub-classes with different complexity and proteaseresistance have previously been identified in inclusion

bodies and also in that case a protein fraction wasstill active. In this study, the structural hetereogenityof the proteins trapped in the aggregates isconfirmed by our data.

Biophysical characterization of the GFP-GST fractionsseparated by the sucrose gradient

The separation of the recombinant GFP-GST on thesucrose gradient is an indication of a mass differenceamong the aggregates and we wished to confirmthese data by size exclusion chromatography (SEC).First, the GFP-GST proteins affinity purified from thefour sucrose gradient fractions were dialysed andanalysed in the fluorimeter according to the methodproposed by Nominé et al., namely the absorbance at280 and 340 nm was measured and the ratiocalculated. This value (aggregation index) indicatesthe relative aggregation, is quickly determined, and

allows the comparison of different fractions of thesame protein. Low values indicate a loweraggregation state and our data show that there is agradient of increasing aggregation from the topfraction to the bottom fractions .

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The 4 GFP-GST fractions were also subjected to SECand the ratio between the areas of the peakscorresponding to the monodispersed and theaggregated protein was calculated (SEC index). Such

an index confirmed an increasing state of aggregationfrom sucrose fraction 1 to 4. Surprisingly, the SECexperiments showed that both aggregated andfunctional forms of the fusion protein were present inboth the three fractions corresponding to theinsoluble GFP-GST and the (soluble) fraction 1.Soluble aggregates have been described before andare probably common when fusion proteins areexpressed. It was not possible to separatemonodispersed GFP-GST from soluble aggregates by

means of sucrose gradients of decreasingconcentrations (data not shown).

We finally tried to characterize the aggregatesaccording to their specific structure. ThioflavinT (ThT)is a dye that preferentially binds to amyloid-likefibrils. We measured an increasing binding whenaggregates of higher complexity were used . Incontrast, there was not significant binding of any

aggregate to 8-anilino-1-naphtalenesulfonic acid(ANSA) that has been used as a marker of theamorphous aggregates . This suggests that theaggregates formed by GFP-GST probably have aregular structure involving β-sheets rather than beinga chaotic complex held together by hydrophobicinteractions. Instead, a micellar organization hasbeen proposed for the soluble aggregates.

Aggregate identification by electron microscopy

In the case of the GFP-GST fractions we showed thatthe degree of amyloidation detected by ThT-bindingprogressively increased from fraction 1 to fraction 4 .The capacity to form fibrils is sequence specific and itseems a generic feature of polypeptide chains. Thedevelopment into fibrils is characterized by a log

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phase during which the aggregation seeds are formedfollowed by a period of rapid growth. Once formed,the fibrils act as aggregation seeds, speeding up theprocess. Therefore, it could be expected that larger

aggregate networks have the possibility to developfaster into structures of higher complexity. In order totest this hypothesis, the GFP-GST from the foursucrose gradient fractions was recovered immediatelyafter centrifugation and mounted for electronmicroscopy analysis.

Some aggregation seeds (20-40 nm in diameter) werevisible even when the GFP-GST from the upperfraction was used (Figure 2A, fraction 1).

Sort of chains composed by globular elementarystructures and measuring several hundreds of nmwere observed when GFP-GST from the fraction 2 wasexploited (Figure 2A) while protofilaments and higherordered fibrils [28] longer than 1 μm (Figure 2A) werevisible when samples from fractions 3 and 4 wereused. Therefore, it was possible to demonstrate therelation between the biochemical indexes used tocharacterize the aggregation of GFP-GST and themacro-aggregation complexity visible by electronmicroscopy.

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Fibrils are the end product of GFP-GST aggregationbut the different classes of aggregates separated bysucrose gradient can be considered as dynamicintermediates that can either develop to larger

structures or be reversed into lower-complexityaggregates. Both the initial complexity and theincubation time of polypeptides prone to aggregationare crucial for the building of the aggregates. Wewished to demonstrate the importance of thesefactors in a control experiment. GFP-GST wasseparated into fractions by sucrose gradient and thefractions 1 and 4 were mounted for electronmicroscopy only after 24 hours of incubation in thepresence of the co-migrated cell components. Both

samples raised similar large fibrils (Figure 2B),indicating that the incubation period was sufficientfor both, independent of their initial aggregationstate, to reach the rapid growth phase that leads tothe fibril formation.

This experiment underlines once more the importanceof the parameter time in studies dealing withaggregation and questions the meaning of some in

vitro experiments. In fact, the fibril maturationoutside the bacterial cell could have peculiarfeatures. For instance, the lack of space-constrain orlimitations in the disaggregation processes couldenable the formation of fibrils the length of which aredifficultly compatible with the size of E. coli cells(Figure 2B). The experiments described in the twolast paragraphs will show the impact of cellcomponents in promoting aggregation anddisaggregation.

Finally, the presence of aggregation seeds smallerthan 40 nm in diameter shows that it is not possibleto discriminate between soluble and aggregatedfractions by the use of simplified methods in high-

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throughput protocols as, for instance, theexploitation of a 0.65 μm pore size filter.

Is the aggregation of GFP-GST actively supported?

In the previous experiments we showed that even themoderately aggregated GFP-GST recovered from theupper fraction of the sucrose gradient could formfibrils if the sample was incubated with the cellfraction for at least 1 day before it was prepared forthe electron microscopy analysis. In a recent paper itwas claimed that bacterial chaperones play an activerole in the formation of the aggregates. The possibleparticipation of cell components in catalyzing the

GFP-GST fibril formation was investigated in a controlexperiment. The process of aggregate maturation of the soluble recombinant protein in the presence of other cell components was limited to 1 hourperforming the affinity purification of the GFP-GSTimmediately after lysis to avoid a seeding processduring the 15 hour centrifugation of the cellcomponents upon the sucrose gradient. The samplewas incubated at room temperature for 4 weeks andthe modifications of the secondary structure weremonitored by CD while corresponding samples weremounted for electron microscopy. No significantmodification was observed in the first two weeks anda slight increase of the β-sheet content was measuredonly after 4 weeks (Figure 3).

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The use of different protein concentrations and theaddition of sucrose to the proteins did not modify thepattern and no detectable aggregate was observed atthe electron microscopy using the correspondingsamples (data not shown).

Therefore, these results strongly suggest that the co-presence of other molecules is necessary to triggerthe process of regular aggregation of the

recombinant protein, probably by facilitating theformation of aggregation seeds. Chaperones can playa role in the aggresome formation and GroEL hasbeen claimed to be actively involved in bacterialinclusion body formation. Our data can only confirmthat GroEL co-migrates with the aggregates of largermass (Fig. 1B). Finally, we are looking for ananalytical method to determine if the process of celllysis is crucial for the development of the aggregates.

Aggregate complexity and re-folding

Both in vivo and in vitro experiments illustrated theco-operative action of chaperone networks indisaggregating misfolded proteins but the features of the real aggregates that are the target of thechaperones in the cells have never been investigated.

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We used the aggregates from fractions 3 and 4 to testif they could be a substrate for chaperone-dependentrefolding and if the different structure complexity hada role on the refolding kinetic.

An equimolar combination of DnaK, DnaJ, GrpE, andClpB quickly disaggregated the large precipitates(Figure 4).

Specifically, the complexity of the aggregates fromfraction 3 was reduced in a faster and more efficientway. In fact, the aggregation index dropped by half in

only 4 min while it took 10 min in the case of theaggregates from fraction 4. Furthermore, there was ahigher residual aggregation: the aggregation indexesmeasured were 1.2 and 0.7 for the aggregates fromfractions 4 and 3, respectively. In comparison, theGFP-GST from fraction 1 scored 0.38. The addition of equimolaramounts 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 isreminiscent of previous works indicating that specificsubclasses of the proteins trapped in the inclusionbodies are preferentially refolded under physiologicalconditions and that the reversibility is increasinglydifficult and dependent on the size of the aggregates.

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The limit of this experiment is that it is difficult toscale up and the small amount of the protein usedwas insufficient for undertaking further biophysicalanalysis. The aggregation index gives only relative

values and, therefore, we can state that the degree of aggregation decreased but cannot conclude that thedisaggregated protein was also correctly folded.Nevertheless, the results suggest that it would be of biotechnological interest to separate the aggregatesubclasses and use the lower complexity aggregatesin refolding protocols.

Conclusion

There is increasing evidence that aggregates areheterogeneous in size and complexity . Theaggresomes are actively built in eukaryotic cells andthe physiological meaning of the process would bethe packing of disorganized aggregates that couldinterfere with the normal cell functions by non-specifically binding to other cell components. Thepossibility to recover functional proteins from theinsoluble aggregates [3] would indicate that at leastin bacteria they can function as a reserve in dynamicequilibrium with soluble fractions.

The expression of recombinant proteins is a stressfactor because they compete for energy andsubstrates with native expression and can interferewith the normal metabolism by forming aggregates,both in prokaryotic and eukaryotic cells. Thepossibility to store the excess of misfolded

recombinant protein could be a way to get rid of dangerous aggregating material when misfoldedproteins escaped the quality control of chaperonesand proteases . The cellular mechanisms that favorthe generation of amyloids (Figure 2) might also beuseful in preventing amorphous aggregates in non-specifically trapping native proteins. The aggregate

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organization would consider an aggregate mash thatgrow from small entities towards larger insolublestructures composed by a core of protease-resistantfibrils, homologous proteins at different levels of

misfolding and some heterologous and non-specifically trapped proteins (Figure 5B).In this paper we present data supporting the idea of aprogressive maturation of recombinant GFP-GSTaggregates into amyloid fibrils. Furthermore, it seemsthat the process is facilitated by some other cellcomponents since the fibril maturation was extremelyslower when the recombinant protein was separatedfrom the other cell components soon after the lysis(Fig. 3). For instance, GroEL has been reported having

an active role in inclusion body formation andspecifically co-migrate with the larger aggregatescould (Fig. 1B). Conversely, the combination of DnaK,DnaJ, GrpE and ClpB could disaggregate largeinsoluble structures (Figures 4 and 5A).

It seems that the aggregation process of recombinantproteins is extremely more complicated than normallyaccepted and our separation protocol turned out to

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be a useful tool for characterizing the aggregates.Furthermore, such an aggregation process sharesmany features with the maturation of pathologicalamyloids in eukaryotic cells and, therefore, the

bacterial system -experimentally easy to modify-would be considered as a model to integrate theresults obtained using in vitro systems and to studythe impact of chemical and biophysical parameters onthe aggregation development. We simplified the work by using a fluorescent construct but any protein forwhich antibodies are available could be used forfollowing the aggregation development.

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b.tech. Biotechnology Notes (6)