Biotechnology Letters 24: 1939–1944, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Protein aggregated into bacterial inclusion bodies does not result inprotection from proteolytic digestion

Xavier Carbonell∗∗ & Antonio Villaverde∗Institut de Biotecnologia i de Biomedicina and Departament de Genetica i de Microbiologia, Universitat Autonomade Barcelona, Bellaterra, 08193 Barcelona, Spain∗∗Present address: Department of Biological Sciences, Gilbert Building, Room 304 385, Stanford University,Stanford, CA 94305-5020, USAAuthor for correspondence (Fax: +34 935812011; E-mail: avillaverde@servet.uab.es)

Received 1 August 2002; Revisions requested 2 September 2002; Revisions received 17 September 2002; Accepted 18 September 2002

Key words: protein aggregation, protein folding, proteolytic cascade, recombinant protein, tailspike protein

Abstract

Proteolytic resistance, as conferred by protein aggregation into inclusion bodies, has not been explored in detail.We have investigated the eventual digestion of several closely-related proteins, namely six insertional and twofusion mutants of the homotrimeric bacteriophage P22 tailspike (TSP) protein. When over-produced in E. coli, allthese polypeptides form inclusion bodies accompanied by only traces of soluble protein. The mutations introducedin TSP impaired its degradation and enhanced its half live up to ten-fold, without affecting protein solubility. Thisindicates that protein properties other than solubility, are the main determinants of susceptibility to proteolysis. Inaddition, the analysis of the degradation fragments strongly suggests that the aggregated TSP polypeptides undergoa site-limited proteolytic attack, and that their complete digestion occurs through an in situ cascade cleavageprocess.

Introduction

In Escherichia coli, the expression of recombinantgenes at high transcription rates usually results inmisfolding of the encoded protein. Chaperones andproteases act as the main cellular controllers of proteinquality (Wickner et al. 1999) by preventing aggre-gation and by promoting protein folding and medi-ating the proteolysis of folding-recalcitrant protein(Schlieker et al. 2002). An incomplete success ofthese activities results in the accumulation of partiallyfolded or misfolded protein chains as inclusion bod-ies (IBs) (Marston 1986). When the synthesis of therecombinant protein is arrested, a significant fractionof the IB protein is removed and re-enters the controlquality network for further refolding or degradation(Carrió & Villaverde 2002).

Proteolysis and aggregation have been often ob-served as exclusive events. In cells devoid of the mainproteases, such as Lon, aggregation of over-produced

proteins is enhanced (Corchero et al. 1996, Rosenet al. 2002). In vivo protein aggregation would then bethe result of misfolded protein escaping from degrada-tion, either by an intrinsic high proteolytic resistanceor because of the under-titration of cell proteases.On the other hand, the stimulation of IB formationby adjusting culture conditions has been suggestedas a strategy to stabilize a given protein against theproteolytic attack (Hellebust et al. 1989). However,several lines of evidence indicate that protein aggre-gation as IBs does not represent a complete protectionfrom proteolysis. In vitro, the controlled digestion ofIBs by incubation with proteases has been used toanalyse the molecular organization of IBs (Bowdenet al. 1991, Carrió et al. 2000). In vivo, protein isremoved from IBs for further refolding or degradation(Carrió & Villaverde 2001), and insoluble polypep-tides in loose aggregates are sensitive to site-limiteddigestion (Corchero et al. 1997). In partially solubleIB-forming proteins, proteolysis can take place during

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the transit of recombinant protein between soluble andinsoluble cell fractions (Carrió et al. 1999). Althoughit cannot be excluded that digestion can also occurin IB-associated polypeptides, the in situ susceptibil-ity of IB protein to degradation remains unexplored.To address this question, we have here investigateda set of engineered P22 tailspike proteins that, uponproduction in E. coli, only occur as IBs without any ac-companying soluble protein. Under these conditions,a cascade of proteolysis has been identified leadingto complete protein digestion, and some degradationintermediates, accounting for more than 50% the totalrecombinant protein, remain trapped in the IBs. On theother hand, the engineering of this protein results in upto a ten-fold variation of its half-life without affectingits solubility, indicating that the soluble form is notthe target of the proteolytic attack. In summary, theseresults indicate that IB formation does not protect thetarget protein from cell proteases and strongly sug-gest that IB polypeptides are vulnerable to an in situsite-limited attack, that is responsible for a cascadedegradation process.

Material and methods

Proteins

Protein rTSP, encoded by plasmid pTTSP (Carbonell& Villaverde 1996), is a recombinant version of TSPof bacteriophage P22, which contains few additionalamino acid residues at the animo (Met-Gly-His) andcarboxy termini (Glu-Phe) as a result of the cloningstrategy. Plasmids pTATSP (Carbonell & Villaverde1998a) and pTTSPA (Carbonell & Villaverde 1996)encode proteins ATSP and TSPA, respectively. Thesemutants carry a 23-residue peptide (named GH23)from foot-and-mouth disease virus that is fused ateither the TSP amino- or carboxy-terminal end. Pro-teins TSP144, TSP328, TSP368 and TSP465, en-coded by plasmids pTTSP144, pTTSP328, pTTSP368and pTTSP465, respectively (Carbonell & Villaverde1998a), are TSP derivatives harboring additionaldipeptides accommodated contiguously to residues143, 327, 367 and 464 (Cys-Ser, Cys-Ser, Leu-Gluand Ala-Asp, respectively). These mutations weregenerated by the introduction of PstI restriction sitesin the encoding gene for cloning purposes. TSP465also presents a Tyr464-Ser substitution. TSP368A andTSP465A (encoded by pTTSP368A and pTTSP465Arespectively), carry the GH23 peptide as encoded by

oligonucleotides introduced at the corresponding PstIsites. Further details can be found elsewhere (Car-bonell & Villaverde 1996, 1998a, b). All the encodingvectors derive from the IPTG-inducible expressionvector pTrc99A (Pharmacia) and they were trans-formed in the E. coli B strain BL26 (F−, λ−) dcmompT hsdS gal lon �lacZ for protein production.

Separation of soluble and insoluble fractions andinclusion body purification

For the relative quantification of soluble and insolubleprotein fractions, IBs were purified as follows. Bacter-ial cultures (30 ml) were grown in LB and plasmid-directed gene expression was induced for 3 h with1 mM IPTG. They were then centrifuged at 4000 × gand 4 ◦C for 15 min and cells were resuspended in 5 mlPBS (phosphate-buffered saline: 2.5 mM NaH2PO4,7.5 mM Na2HPO4, 0.14 M NaCl), washed once inthis buffer and finally resuspended in 3 ml lysis buffer(50 mM Tris/HCl, 1 mM EDTA, 100 mM NaCl, pH 8)per g of wet cells. After freezing and thawing, 4 µl100 mM PMSF in 2-propanol per g pellet and 80 µllysozyme per g pellet (10 mg ml−1) were added. Theresulting mixture was warmed for 45 min at 37 ◦Cwith gentle shaking. NP-40 (non-ionic detergent P-40) was then added to give 1% (v/v) and sampleswere then further kept at 4 ◦C in mild agitation for45 min. Afterwards, the mixture was supplementedwith 10 mM MgSO4 and DNase I to 25 mg ml−1

(final concentrations). After incubating at 37 ◦C for45 additional min, samples were centrifuged for 15min at 12 000 × g and 4 ◦C and supernatants keptfor further analysis. The insoluble fraction was furtherwashed in 9 vols. of washing buffer [50 mM Tris/HCl,10 mM EDTA, 100 mM NaCl, 0.5% (v/v) Triton X-100, pH 8] and resuspended in 1.5 supernatant vols. of1× loading buffer [62.5 mM Tris/HCl, 3% SDS, 10%glycerol (v/v), 715 mM 2-mercaptoethanol and 0.03%Bromophenol Blue, pH 6.8] plus 4 M urea. To super-natants, 0.5 vols. of 2× loading buffer plus urea wereadded. All samples were boiled for 10 min, loaded in7.5% (w/v) polyacrylamide gels and ran for 45 minat a constant voltage of 200 V. Proteins were thenelectrotransferred to nitocellulose membranes and im-munodetected with a rabbit anti-TSP serum. Proteinbands were developed by a goat anti-rabbit IgG an-tibody coupled to horseradish peroxidase and using4-chloro-1-naphthol and H2O2 as substrates as indi-cated. After scanning, band intensity in both solubleand insoluble protein fractions was quantified with Bio

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Image Intelligent Quantifier 2.5.0 (Bio Image SystemsCorp.).

Analysis of protein degradation

For the study of intact and degraded protein fractions,1 ml samples were withdrawn from BL26 culturesat intervals. Cells were collected by centrifugation,resuspended in loading buffer to a final OD550 of 4(1.3 g l−1 cell dry wt) and boiled for 10 min. Eachsample, 20 µl, was loaded in SDS-PAGE and thenelectrotransferred to nitrocellulose membranes. Bandsfrom these crude cell extracts were developed andquantified as stated before. To calculate proteolyticdegradation rates, BL26 cultures were grown on LBmedium up to an OD550 of 0.4 (0.13 g l−1 cell drywt), and then expression of recombinant TSPs wasinduced by adding IPTG at 1 mM. Protein syntheseswere stopped 1 h later by adding chloramphenicol at200 mg l−1. SDS-PAGE and Western blot were per-formed as described above. Densitometric data werefitted to an exponential decay equation with SigmaPlot2000 (SPSS Inc.) to obtain half-life values.

Results

Solubility of TSP and TSP variants

Protein rTSP and all its engineered derivatives pro-duced clear inclusion bodies (IBs) in bacterial culturesupon addition of IPTG (not shown). The eventual pres-ence of soluble forms was determined by the TSPamounts in both the soluble and insoluble cell frac-tions. Under the stated gene expression conditions, thesoluble protein represented between 0.03 and 0.3% ofthe total recombinant protein for rTSP, TSPA, ATSPand TSP144, and it was much lower (in some casesbeing undetectable) for the rest of TSP mutants (Ta-ble 1). Although from this data it cannot be completelyexcluded that the performed insertional mutagenesismight have slight effects on TSP solubility, its possibleimpact would range within an already low percentageof remaining soluble protein, below 0.3%.

TSP protein accumulation upon gene expression

When analysing production of recombinant proteins(Figure 1), higher amounts of TSP variants were ob-served when compared with the parental, pseudo wildtype rTSP, only TSPA-producing cells yielding similarlow recombinant protein levels. Since the expression

Table 1. Solubility of engineered tailspike proteins.

Proteina Cell fractionb Soluble form

Soluble Insoluble (%)

rTSP 6.13 × 10−3 2.06 0.299

ATSP 1.4 × 10−3 1.03 0.136

TSPA 8.75 × 10−4 2.68 0.033

TSP144 9.7 × 10−3 10.96 0.088

TSP328 n.d. 59.18 <4.22 × 10−5

TSP368 n.d. 24.46 <1.02 × 10−4

TSP465 1.13 × 10−3 13.85 0.008

TSP368A n.d. 5.51 <4.54 × 10−4

TSP465A n.d. 5.22 <4.79 × 10−4

aDetails of all these proteins can be found elsewhere (Car-bonell & Villaverde 1998a).bThe integrated intensity of each intact band is given in densit-ometric units. Samples were obtained from cultures 3 h afterIPTG addition.n.d. – Not detected. The detection limit in this analysis is 2.5× 10−5 densitometric units.

vector and production conditions were the same inall the cases, the yield differences might result froman enhanced resistance to proteolysis as promoted byTSP engineering. This possibility was investigated bycalculating the in vivo half-lives of the selected modelproteins rTSP, TSPA and TSP465A in E. coli cells.Protein TSP465A was indeed extremely stable (halflife of 1732 min) in opposition to rTSP and TSPA,exhibiting half-lives of 141 min and 89 min, respec-tively. Therefore, the different productivity of theseTSP variants was indeed connected to distinguishabledegradation rates. Noteworthy, the observed variationsin the proteolytic stability occurred without majormodifications in the solubility of proteins (Table 1).

Analysis of degradation intermediates

Interestingly, proteins rTSP and TSPA, being rapidlydegraded, were only observed in the IB as the in-tact, full-length forms, while in the rest of IBs,the intact form was accompanied by varying pro-portions of degradation intermediates (Figure 1, Ta-ble 2). TSP465A IBs were formed by about 50%degradation intermediates versus about 15% for rTSP(while TSP465A was 10 fold more stable than rTSP).Therefore, the degradation intermediates from a givenrecombinant protein do not necessarily indicate anenhanced proteolysis as generally assumed. Contrar-ily, it might be the result of bottlenecks arising inan impaired digestion process, during which stabledegradation intermediates accumulate in the cell.

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Fig. 1. Recombinant tailspike proteins upon IPTG-mediated gene expression as detected by Western blot of cell extracts. Figures indicate thetime (in h) after the addition of IPTG. Time 0 was sampled immediately before IPTG addition. In lane (−) a culture aliquot was separatedbefore induction and cultures kept under the same conditions without IPTG for 4 h. Molecular weight markers were run in the lane (m).

In addition, the introduced mutations resulted inthe accumulation of digestion fragments representingup to 90% of the total IB protein. Again, since theamount of soluble protein was extremely low and notsignificantly affected by mutations, the protein, aspresent in the soluble cell fraction, is not requiredby the accumulation of such fragments. This stronglysuggests that the generation of such fragments canonly take place on already insoluble, IB-associatedprotein. On the other hand, the analysis of the degrada-tion intermediates found in most of the TSP-based IBsshow a coincident degradation pattern. For instance,

a protein species migrating as twin bands of about60 kDa abounds and also a minor population of het-erogeneous polypeptides of about 50 kDa (Figure 2)were occurring in all the proteins in which degrada-tion intermediates were observed. This concurrence ofdegradation intermediates from different TSP mutantsindicates a defined cascade process for TSP degrada-tion by sequential site-limited cleavage as observedin other recombinant, engineered proteins (Corcheroet al. 1997). This cascade process is probably occur-ring also during the degradation of rTSP and TSPA,but not noticed because of the high velocity of these

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Table 2. Full-length tailspike protein in total cell extracts.

Protein Intact proteina (%)

0 hb 1 h 2 h 3 h 4 h 24 h (−)

rTSP n.d. 79.8 95.9 88.1 89.7 85.7 31

TSP144 76.7 46.1 54.4 54.3 50.1 82.8 94.6

TSP328 87.8 64.6 58.9 41.6 69.3 53 90.5

TSP368 93.8 80.4 79.2 81.4 75.9 77.5 92.3

TSP465 100 61.3 59.3 55.6 57.5 56.7 74.8

ATSP 100 10.1 19.3 19.8 33.8 38.3 95.9

TSP368A 100 42.5 35.2 51.6 46.3 47.1 84.1

TSP465A 100 55.3 51.3 58.6 49.8 49.1 90.8

TSPA n.d. 100 100 100 100 100 n.d.

aThe integrated intensity of intact TSP bands (in densitometricunits) relative to the total integrated intensity of the corre-sponding lane, including all the degradation bands.bSamples were withdrawn immediately before IPTG addition(time 0 h), and at different times after induction of gene expres-sion. At time 0 h, aliquots of the cultures were kept withoutIPTG and cultured under the same conditions for 4 h (−).n.d.– The full-length TSP band was not detected in thesesamples.

processes. Therefore, the observation of fragmentsfrom these protein variants would result from theirimportant stabilization by the mutations introduced inTSP. It must be stressed that these intermediates wereonly observed in purified IBs but not in the minorsoluble fraction.

Discussion

Bacterial IBs are refractile protein aggregates usu-ally occurring in recombinant bacteria. Recent studieshave revealed that IBs are not the dead-end of in-correct folding pathways but transient reservoirs ofloosely packaged folding intermediates, deeply inte-grated in the cell quality control system (Carrió &Villaverde 2002, Schlieker et al. 2002). In absence ofrecombinant protein synthesis, IB protein is efficientlyremoved and refolded but an important fraction is de-graded (Carrió & Villaverde 2001). In partially solubleproteins, the presence of degradation fragments in IBsresults at least in part, from re-aggregation of digestionproducts generated in the soluble cell fraction (Carrióet al. 1999). However, it is not yet clear if insolublepolypeptides are also sensitive to in situ proteolyticattack as associated to IBs.

In the present study we have observed a cascadedigestion process and ten-fold variations in the rateof protein degradation in a set of related IB recombi-

Fig. 2. Band migration analysis of tailspike proteins and theirdegradation intermediates. This analysis was done on samples taken3 h after IPTG addition.

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nant proteins. Since the soluble form of these proteinsis hardly detectable (Table 1), this indicates that theproteolytic stability of IB proteins is not (at leastsignificantly) depending on their solubility. In thiscontext, minor variations in the already low solublefraction (always less than 0.3%) found between someproteins do not have a direct influence on the digestionrate. For instance, rTSP and TSPA are present in thesoluble cell fraction in 0.299 and 0.033% of the totalprotein respectively (a ten-fold difference), while theirhalf-lives are hardly differing by two-fold (141 minand 89 min respectively).

On the other hand, the presence of a soluble pro-tein fraction is not required for a cascade proteolyticdigestion since the soluble forms of TSP328, TSP368,TSP368A and TSP465A mutant proteins are not de-tected, while coincident degradation fragments occurin the respective IBs (Figure 1). These fragments rep-resent an important fraction of the total IB protein(Table 2). In addition, TSP engineering can then im-pair their turnover (as proved by the extended half-lifeof TSP465A) by stabilizing some degradation inter-mediates (Figure 1). However, rTSP is more rapidlyproteolysed even in absence of detectable degradationintermediates. This indicates that the observation ofsuch fragments, a common fact during recombinantprotein production, can eventually indicate a slow,impaired degradation process rather than a high pro-teolytic sensitivity as generally assumed. Then, theseresults prove that polypeptides aggregated as IB canundergo a cascade proteolytic attack resulting in asequential digestion process, for which the solubleform of the protein is not required. This stronglysuggests that this site-limited, sequential cleavage oc-curs in situ on IB-associated polypeptides withoutimmediate release of the digestion fragments.

Acknowledgements

This work has been supported by grant BIO2001-2443 (CICYT) and by the Ma Francesca de RoviraltaFoundation and it has been partially written at theGBF (Germany) under a grant from the DeutscheForschungsgemeinschaft (SFB 578 ‘Vom Gen zumProdukt’) to AV.

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