497
`
`Advancesin refolding of proteins produced in E. coli
`HaukeLilie, Elisabeth Schwarz and Rainer Rudolph*
`
`Inclusion body production is a common themein recombinant
`protein technology. Hence, renaturation of these inclusion
`body proteins is a field of increasing interest for gaining large
`amounts of proteins. Recent developments of renaturation
`proceduresincludethe inhibition of aggregation during
`refolding by the application of law molecular weight additives
`and matrix-bound renaturation techniques.
`
`Addresses
`Institut fur Biotechnologie, Martin Luther Universitat Halle-Wittenberg,
`Kurt Mothes Strasse 3, D-06120 Halle, Germany
`*e-mail: rudoloh@biochemtech.uni-halle.de
`
`Current Opinion in Biotechnology 1998, 9:497-501
`
`http://biomednet.com/elecref/0958 166900900497
`
`© Current Biology Ltd ISSN 0958-1669
`Abbreviation
`GdmCl_
`guanidinium hydrochloride
`
`Introduction
`Elegant and well established recombinant DNA method-
`ologies have set
`the stage for
`the production of
`heterologous proteins in microbial bosts. The abundance
`of protein expression systems renders the efficient bacter-
`ial production of most proteins possible; however, high
`level expression of recombinance protein often results in
`accumulation in
`bodies.
`aggregation and
`inclusion
`Deposition of the recombinant protein in inclusion badies
`can be heaven or hell. The latter applies to those protcins
`for which renaturation 1s problematical. Here, the only way
`out is the avoidance or at least reduction of inclusion body
`formation. In contrast, in the case of a simple andefficient
`renaturation procedure, deposition of the protein in inclu-
`sion bodies and subsequent isolation and renaturation of
`inclusion bedy protein often means the most straightfor-
`ward strategy to get
`large amounts of the recombinant
`protein.
`In this review we present an extract of recent
`developments in inclusion body refolding.
`
`disulfide bonds does usually not occur in this reducing cel-
`Jular compartment. Phe consequence is improperfolding
`resulting in aggregation.
`
`An increase in the concentration of non-native polypep-
`cides due to high expression levels seems to be responsible
`for aggregation of the recombinant protein. ‘Vhis assump-
`don was quantified in a kinetie model that analysed the
`vield of native protein as a funetion of the competition
`between folding and aggregation [2]. Aceording to this
`model, the relative yield of native protein increased with a
`decreased rate of protein synthesis. Qualitatively, this was
`confirmed by recombinant protein expression at optimal
`and subopcamal conditions, Thus, whereas recombinant
`proteins often aggregate when Aseherihia cofi cells are cul-
`tivated at 37°C, reduction of che cultivation cemperature
`can increase the amount of native protein due to a
`deercase of the ratte of protein synthesis [3].
`
`the addition of non-metabolizable carbon
`Alternatively,
`sources, such as desoxyglucose, at che time of induction
`leads to a reduced metabolie rate, which results in a limitc-
`ed production of the recombinant protein. Another
`possibility is limited induction of gene expression by the
`promotors, which can be linearily rezulated by the induc-
`er concentration, Recombinant protein deposition in
`inclusion bodies is commonly observed with hydrophobic
`proteins.
`Ilere,
`fusion with a hydrophilic protein can
`enhance solubilizy. Widely used fusion partnerproteins are
`elutathione-S-ctransferasc, malctose-binding protein or
`thioredoxin [4,5].
`
`A wayof taking advantage of the host ccll’s equipment ta
`deal with protem folding is the co-overexpression of the
`recombinant protein with molecular chaperones [6,7°,8,9}.
`Sull, che beneficial effect of co-overexpression af chaper-
`one proteins
`is
`unpredictable,
`as
`the
`appropriate
`substrate-chaperone combination is a matter of trial and
`error [10}.
`
`Inclusion body formation
`inclusion
`Upon overexpression of recombinant proteins,
`bodies can be observed in several host systems, for exam-
`ple, prokaryotes, yeast or higher eukaryotes, Even
`endogenous proteins, if overexpressed, can accumulate in
`inclusion bodies [1]. suggesting chat in most cases inclu-
`sion body formation is a consequence of high expression
`rates, regardless of the system or protein used. There is no
`direct correlation between the propensity of inclusion
`body formation of a certain protein and its intrinsic prop-
`ertics, such as molecular weight, hydrophobicity, folding
`pathways, and so on, Onlyin the case of disulfide bonded
`proteins can inclusion bodyformation be anticipated if che
`protein is produced tn the bacterial cytosol, as formation of
`
`In the case of disulfide-containing proteins, the principles
`mentioned above are not sufficient to avoid inclusion body
`formation. Although the reducing conditions of the bacte-
`rial cytosol allow disulfide bond formation in a few cases
`[11], disulfides are not normally formed in this compatt-
`ment.
`In contrast,
`the prokaryotic periplasm provides
`oxidizing conditions for disulfide bonding; however, inclu-
`sion body formation can also occur in the periplasm [12].
`In particular, the native periplasmic expression ofproteins
`that possess several disulfide-bonds is problematical as the
`endogenous periplasmic enzyme disulfide bond oxidore-
`ductase (DsbA) merely introduces disulfides into proteins,
`but dees not catalyze disulfide reshuffling, A means to
`enhance correction of incorrect disulfide-bonds in the
`Amgen Exhibit 2031
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Pace 1
`
`Amgen Exhibit 2031
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`

`

`498 Expression vectors and delivery systems
`
`Figure 1
`
` optimized fermentation protocol can decrease degradation
`
`
`of the recombinant product. A shift to elevated tempera-
`tures (~42°C) at
`the time of induction to accelerate che
`aggregation process further decreases protcalysis.
`
`Preparation and solubilization
`‘The basic principle of inclusion body preparation is a liq-
`uid/solid separation. Because inclusion bodies have a
`relatively high density (~1.3 mg/ml [18]) they can be pel-
`leted by centrifugation. It
`is importance that cell
`tysis is
`complete, because intact cells sediment together with the
`inclusion bodies, chus, contaminating the preparation with
`host protein. “he most etfcetive procedure for complete
`cell disruption is a high pressure dispersion following a
`lysozyme treatment. A further treatment of the crude
`lysate with detergents solubilizes lipids and membrane
`proteins (for protocol see [19°]). In a few cases, urea or
`guanidinium hydrochloride (GdmCl) at low concentrations
`can dissociate inclusion body associated proteins. It should
`be kept in mind, however, that a too high urea or GdmCl
`concentration will lead co the solubilization of the inclu-
`sion bodies themselves. On average, an inclusion bady
`preparation contains more than 50%of the recombinant
`protein, the purity of the preparation may reach up to 90%
`under optima] conditions.
`
`Before starting renaturation the inclusion body material
`has to be solubilized by strong denaturants, such as 6 M
`GdmGl or 8 M urea. The addition of dithiothreitol keeps
`all cysteines in the reduced state and eleaves disulfide
`bonds formed during preparation. Before renaturation dial-
`ysis against GdinC] at low pH without reducing agents can
`be performed to remove the dithiothreitol for subsequent
`storage (for protocol sce [19*]).
`
`Renaturation of inclusion body proteins
`Renaturation ofsolubilized inclusion bodies is initiated by
`the removal of the denaturant either bydialysis or dilution.
`As discussed for the 7# veo situation,
`the efficiency of
`renaturation depends on the competition between correct
`folding and aggregation (Figure 2). Ageregation of the non-
`native recombinant protein may be enhanced by other
`protcins contaminating the inclusion body preparauon if
`they themselves tend to aggregate [20°].
`
`To slow down the aggregation process refolding is usually
`performed at
`low protein concentrations,
`in a range of
`10-100 pg/ml. Furthermore, renaturation conditions must
`be carefully optimized regarding external parameters, such
`as temperature, pH or ionic strength. Even in an optimized
`system, however, the yield of renaturation maybe relative-
`ly low, necessitating large volumesfor preparation of large
`quantities of the native protein. Because during refolding
`only the concentration af non-native polypeptides and not
`that of native protein ts critical for aggregation, a strategy
`to circumvent
`the problem oflarge refolding vessels ts
`pulse renacuration [21]. In order to keep the concentration
`of the unfolded protein low,
`thus limiting agercgation,
`
`Page 2
`
`E. cof expressing the heavy chain of the antibody MAK 33. The cells
`were harvested six hours after induction of expression. Preparation for
`electron microscopy includedfixation with glutaraldehyde, embedding
`in Epon resin and negative staining with uranyl acetate, The inclusion
`bodies are visible as amorphous light grey structures.
`
`periplasm of /. cof is to overexpress the endogenous
`periplasmic DsbC protein, which is a disulfide isomerase
`[13*,14]. Also, cultivation in the presence of thiol reagents,
`which lead co reshnffling of incorrect disulfide bridges, has
`been proven to enhance the yield of native protcins con-
`taining multiple disulfide bridges [15,P1].
`
`Properties of inclusion bedies
`Inclusion bodies are very dense particles of aggregated
`protcin (Figure 1). Because of their refractile property they
`can be visualized by ight microscopy. Inclusion bodies
`may reach sizes with a diameter in the Wmg range and
`exhibit an amorphous or puracrystalline structure indepen-
`dent of their subcellular
`location. Under appropriate
`conditions the recombinant protein deposited in inclusion
`bodies amaunts to about 50% or more of the total cell pro-
`tein. These inclusion bodies often contain almost
`exclusively the overexpressed protein, Major contaminants
`of inclusion body material after preparation are outer mem-
`brane proteins, which are themselves not part of the
`inclusion body particles but copurify as non-solubilized
`protein with the inclusion body fraction. Separation of
`these membrane proteins from inclusion body material can
`be achieved by extensive washing with detergents.
`
`Little is known about the structural propertucs of rhe
`apgregated protein. Structural analyses of inclusion body
`proteins indicate that
`the aggregates possess a certain
`amount of secondary struccure [16], a result also observed
`for 1 vitro aggregated proteins [17].
`
`Being aggregated polypeptides, inclusion bodies are gencr-
`ally not very sensitive to proteolytic breakdown. Stull,
`degradation produets can often be detected in inclusion
`bodies. After analysis of the expression kinetics, an
`
`Page 2
`
`

`

`Advancesin refolding of proteins produced in &. colf Lilie, Schwarz and Rudalph
`
`499
`
`ghitathione [P2]. The advantageof this procedure is that the
`mixed disulfide form of the protein carries additional
`charged residues provided bythe glutathione moiety, which
`increases the solubilicy of the protein during refolding.
`
`Recent studies on disulfide bond formation within pep-
`tides showed the possibility of a directed reaction.
`Oxidation of peptides containing both two selenocysteines
`and two cysteines leads to the formation of two disulfides:
`one between the selenocysteine residues and another
`between the cysceines. No disufide bond between seicno-
`cysteine and cysteine was observed[25]. “Vhis result was
`independent of the order of selenocysteines and cysteines
`within the peptide. Because the peptide did not possess
`any conformational constraints directing disulfide bond
`formation. these results clearly demonstrate the specific
`reactivity due to che different chemical properties of
`selenocystcines and cystcines. Whether such disulfide
`engineering can be transposed on proteins ta prevent the
`formation of wrong disulfides remains to be seen.
`
`In addition to che control of parameters such as tempera-
`ture, pH] or
`redox conditions,
`the presence of low
`molecular weight compounds in the renaturation buffer
`may prove to have a tremendous effect on the yield of
`renacuration [26,27]. Specific cofactors, such as Zn2* or
`Ca2+, can stabilize proceins already at the level of folding
`intermediates,
`thus, preventing off-pathway reacttons.
`Besides such cofactors and prosthetic groups, a large series
`of low molecular weight additives are, in certain cases, very
`efficient folding enhancers: non-denaturing concentrations
`of chaotrophs, such as urea or GdmCl, for example, are
`essential for the renaturation of reduced chymotrypsino-
`gen A and have been shown to promote folding of several
`other proteins [P3]. On the other hand, by slowing down
`the refolding kinetics, GdmC] and urea can shift the com-
`petition between renacuration and ageregarion towards the
`agerepation reaction,
`
`A popular additive is L-arginine [28-30]. In the case of a
`truncated form of plasminogen activator, the yield of renat-
`uration is abour 80% in the presence of 0.5 M L-arginine,
`whereas in the absence ofthis additive almost no reactivi-
`ty was observed [P2]. Vhe mechanism by which L-arginine
`supports renaturation ts still unknown. [.-arginine slightly
`destabilizes proteins [31] in a manner comparable cto low
`concentrations of chaotrophs,
`"The benefical effect of
`|.-arginine on refolding, however, probablyoriginates from
`an increased solubilization of folding intermediates.
`
`LDH
`
`* E
`
`ffect of protein concentration on the renaturation of lactate
`dehydrogenase (taken trom [18]}. Acid denaturedlactate
`dehydrogenase was renatured in 0.1 M phosphate buffer pH 7, 1 mM
`EDTA, 1 mM DTE, 20°C.After 192 hours reactivation (Q) and
`aggregation (A) were quantified.
`
`aliquots of denatured protein are added at defined ume
`points to che renacturation buffer.
`‘Vhe time interval
`between two pulses has to be optimized acvording to the
`refolding and aggregation behaviour of the respective pro-
`tein. The pulse renacuration is stopped when the
`concentration of denaturant,
`tntroduced in the refolding
`buffer together with the denatured protein, reaches a crit-
`ical level, that is, a concentration at which even the native
`protein devclops the propensity to aggregate.
`
`If proteins contain disulfide bonds, the renaturation buffer
`has to be supplemented with a redox system, The addition
`of a mixture of the reduced and oxidized forms of low mol-
`ecular weight thiol reagents, such as glutathione, eysteine
`and cysteamine (molar ratios of reduced to oxidized com-
`pounds
`1:1
`to 5:1,
`respectively), usually provides the
`appropriate redox potential to allow formation and reshuf-
`fling of disulfides [22,23]. The redox system described
`above is characterized by overall
`reducing conditions.
`Correct disulfides are protected by the stable native struc-
`ture; however,
`these correct disulfide bonds within the
`native structure which are still solvent accessible could be
`reduced under these conditions, In such a case, a subse-
`quent strongly oxidizing step using an excess of oxidized
`thiol reagents [24] or copperinduced air oxidation can
`ensure formation ofthe respective disulfide bonds.
`
`For certain proteins, probably due to low solubility of fold-
`ing intermediates, oxidative refolding is not very cftective.
`In chis case, the yield of renaturation may be inercasedif the
`denatured protein is first completely oxidized in the pres-
`ence of a large excess of oxidized glutathione leading to the
`conversion of all SH- groups
`ro mixed disulfides.
`Renaturation of the protein is performed by dilutian ina
`renaturation buffer containing catalytic amounts of reduced
`
`Likewise, increased solubilization of folding intermediates
`can explain the positive effect of detergents on the refolding
`vield. Both ionic and non-ionic detergents can be used to
`suppress ageregation upon dilution of the denatured protein
`in the renacuration buffer. Using laurylmaltosid, Chaps (3-[3-
`chloramidopropyt] dimethylammonia-1-propanc sulfonate)
`or some other detergents during renaturation, che yield of
`refolded protein can be improved [32-34,P4]. Other
`
`Page 3
`
`
`
`
`Figure 2
`
`Cop {ugimt)
`
`
`
`©Reactivation(%e)
`
`
`
`
`
`utOo[>Aggregalion("f.)
`
`Page 3
`
`

`

`500 Expression vectors and delivery systems
`
`detergents, inhibiting renaturation, have to be removed after
`dilution by addition of evclodextrin, which specifically binds
`detergent molecules [35].
`
`Another possibility for suppressing unspecific intermolecu-
`lar interactions is the coupling of the denatured protein toa
`matrix, Denatured G-glucosidase fused to a poly-arginine
`tag was bound to Heparin-Sepharose, Renaruration under
`conditions at which the protein is still bound co the matrix
`resulted in high vields of active protein even at protein con-
`centrations ina mg/ml range (36]. Another matrix used for
`this kind of renaturation was Ni-N’VPA agarose. originally
`developed for an efficient protein purification. After binding
`the denatured protein to the matrix via a ITis-tag the column
`is equilibrated with renaturation buffer and the refolded
`protein can be cluted by an imidazole or pll gradient. Hirst
`demonstrated for chloramphenicol acetyl
`transferase,
`recently, the oxtdacve renaturation of a disulfide bridged
`protein TIMP 3 on a Ni-agarose was deseribed [37,38].
`
`7. Machida 8, Yu Y, Singh SP, Kim J-D, Hayashi K, Kawata Y:
`.
`Overpreduction of §-glucesidasein active form by an Escherichia
`coli system coexpressing the chaperonin GroEL/ES. FEMS
`Microbiol Lett 1998, 159:41-46.
`The expression of recombinant [i-glucosidase with coexpression of the
`GroE system in £ coli was analysed.
`in the absence of GroE,
`the
`recombinant protein was expressed in the insoluble fraction and constituted
`80%of the total cellular protein. The coexpression of GroEL/ES leads to a
`significant fraction of soluble active B-glucosidase, which is even more
`increased at low induction temperatures.
`8.
`Cole PA: Chaperone-assisted protein expression. Siruciure 1996,
`4:939-249.
`
`9.
`
`Thomas JG, Ayling A, Baneyx F: Molecular chaperones,folding
`catalysts, and the recovery of active recombinant proteins from
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`
`10. YasukawaT, Kanei-ishii C. MaekawaT, Fujimoto J, YamamotoT,tshii S:
`Increase of solubility in Escherichia coli by coproductian of the
`bacterial thioredoxin. J Biol Chem 1995, 270:25328-25331,
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`11. Miele L, Cordella-Miele E, Mukherjee AB; High level bacterial
`expression of uteroglobin, a dimeric eukaryotic protein with two
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`/ Biol Chem 1990, 265:6497-6445.
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`12. Georgiou G, Telford JN, Shuler ML, Wilson DB: Localization of
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`Conclusions
`‘Todays detailed knowledge of the mechanisms of protein
`folding and its off-pathwavyreactions, as well as the interre-
`lation of protein structure and folding, makes it possible to
`basically design renaturation experiments. Sell, the specif-
`i¢ conditions
`regarding buffer composition, protein
`concentration, temperature, and so on, has to be optimized
`for every protein. Failure of renaturation may be caused by
`omission of cofactors, such as structurally important metal
`ions, or degradation by traces of proteases. For those pro-
`teins which are synthesized as proforms ## avo,
`the
`prosequence maybe crucial for structure formation and has
`to be inchided in the refolding scheme [39]. Structural and
`16. Oberg K, Chrunyk BA, Wetzel R, Fink AL: Native like secondary
`functional analvses of proteins, especially for therapeutic or
`structure in interleukin-1 beta inclusion bodies by attenuated total
`reflectance FTIR. Biochemisiry 1994, 33:2628-2634.
`other industrial applications, require large amounts of pro-
`1%=Zettimeissi G, Rudolph R, Jaenicke R: Reconstitution of lactic
`teins. Inclusion body production and protein renaturation
`dehydrogenase. Noncovatent aggregation vs. reactivation. 1.
`provides un cfficient route to mect these requirements.
`Physical properties and kinetics of aggregation. Biochemistry
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`
`13, Sone M, Akiyama Y, ko K: Differential in vive roles played by DsbA
`.
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`Using a mutant form of alkaline phosphatase, disulfide band formation in the
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`to the native one. This conversion did net occur in DsbC-disrupted cells. In
`DsbA-disrupted cells, the disulfide formation was less efficient, but
`the
`disulfides formed were predominantly native ones.
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