157
`
`Refolding of recombinant proteins
`Eliana De Bernardez Clark
`
`Expression of recombinant proteins as inclusion bodies
`in bacteria is one of the most efficient ways to produce
`cloned proteins, as long as the inclusion body protein can
`be successfully refolded. Aggregation is the leading cause
`of decreased refolding yields. Developments during the past
`year have advanced our understanding of the mechanism
`of aggregation in in vitro protein folding. New additives to
`prevent aggregation have been added to a growing list. A
`wealth of literature on the role of chaperones and foldases in
`in vivo protein folding has triggered the development of new
`additives and processes that mimic chaperone aclivity in vitro.
`
`Addresses
`Department of Chemical Engineering, Tufts University, Medford,
`MA 02155, USA; e-mail: edeberna@tufts.edu
`
`Current Opinion in Biotechnology 199B, 9:157-163
`
`http://biomednet.com/elecref/095B 1 66900900157
`
`© Current Biology ISSN 095B-1669
`
`Abbreviations
`DTT
`dithiotreilol
`GdmCI guanidinium chloride
`
`Introduction
`Expression of cloned genes in bacteria is widely used
`both in industry, for the production of pharmaceutical
`proteins, and in research, for the production of proteins
`for structural and/or biochemical studies. Bacteria produce
`large quantities of recombinant proteins in rapid, often
`inexpensive, fermentation processes; however, the product
`of interest is frequently deposited in insoluble inactive
`inclusion bodies. The general strategy
`aggregates or
`used
`to recover active protein from inclusion bodies
`involves three steps: firstly, inclusion body isolation and
`washing; secondly, solubilization of the aggregated protein,
`which causes denaturation; and finally, refolding of the
`solubilized protein. While the efficiency of the first two
`steps can he relatively high, folding yields may be limited
`by the production of inactive misfolded species as well
`as aggregates.
`
`When the formation of inclusion bodies was first observed
`almost two decades ago, existing protein folding protocols
`were not, in most cases, applicable to the folding of recom(cid:173)
`binant mammalian proteins, which are in most cases mul(cid:173)
`tidomain, oligomeric, and/or disulphide bonded proteins.
`Existing protein folding protocols had been developed to
`characterize folding intermediates and investigate folding
`pathways of small, monomeric proteins. When applied to
`the refolding of inclusion body proteins, these protocols
`failed to produce active proteins with significant yields.
`Even today, the literature on identification of protein
`
`the elucidation of folding
`intermediates and
`folding
`pathways deals mostly with small monomeric proteins
`that have either intact or no disulphide bonds [I]. For
`many years, cukaryotic expression hosts which produced
`soluble secreted recombinant proteins became favored
`over bacterial hosts because of the difficulties encountered
`when refolding inclusion body proteins; however, careful
`examination of the folding conditions allowed researchers
`to find ways to refold multidomain disulphide bonded
`proteins with relatively high yields. Most of the original
`work on inclusion body protein folding can be found in
`the patent literature starting around 1985 [2].
`
`The recent literature includes many examples in which
`recombinant proteins have been produced by refold(cid:173)
`ing from inclusion bodies. Some of these applications
`demonstrate the use of suboptimal refolding protocols
`to produce small quantities of protein for structural
`and/or biochemical studies. Other applications deal with
`commercial processes. To be acceptable for commercial
`applications, refolding processes must he fast, inexpensive
`and highly efficient. This review focuses on recent
`developments in the optimization of refolding processes
`with emphasis on methodologies applicable to large-scale
`protein production. Since most proteins of commercial
`value are secreted in their natural host and are likely to
`contain disulphide bonds, this review emphasizes recent
`progress in protein refolding with concomitant disulphide
`bond formation, also called oxidative protein refolding.
`
`Inclusion body isolation and solubilization
`Expression of recombinant proteins as inclusion bodies
`can be advantageous due to the very high levels of
`enriched protein produced and the protection of the
`protein product from proteolytic degradation. In addition,
`when producing a recombinant product which, when
`active, can be toxic or lethal to the host cell, inclusion
`body production may be the best available method. Cells
`containing inclusion bodies are typically disrupted by high
`pressure homogenization and the resulting suspension is
`centrifuged to remove the soluble fraction. Occasionally a
`lytic enzyme, such as lysozyme, may be added before cell
`disruption to increase efficiency and reduce power require(cid:173)
`ments. The resulting inclusion body-containing pellet is
`washed with buffers containing either low concentrations
`of chaotropic agents, such as urea or guanidinium chloride
`(GdmCl), or detergents, such as Triton X-100 [3•,4,5•]
`and sodium deoxycholate [4,6,7]. This washing step is
`designed to remove contaminants, especially proteins, that
`may have adsorbed onto the hydrophobic inclusion bodies
`during processing, and could affect protein refolding
`yield. Alternatively, sucrose gradient centrifugation may he
`performed to purify inclusion bodies and separate them
`from other cellular components [4,Pl]. After washing,
`
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`

`158 Biochemical engineering
`
`inclusion bodies arc solubilized using strong denaturants,
`such as urea, GdmCl, or thiocyanate salts, or detergents,
`such as SOS [8°,Pl], n-cetyl trimethylammonium chloride
`[4], sarkosyl [6], or sodium n-laurosyl sarcosine [7], and
`a reducing agent, such as ri-mercaptoethanol, dithiotreitol
`( DTT), dithioerythritol, or cystcinc. Temperatures above
`30'C are typically used tu facilitate the solubilization
`process. A chelating agent, such as EDTA or EGTA, can
`be included in the solubilization buffer to scavenge metal
`ions, which could cause unwanted oxidation reactions.
`Solubilization can also be accomplished by the addition
`of acids, such as 70% formic acid [5•]. Alternatively, for
`periplasmic inclusion bodies the recombinant protein may
`be recovered by in-situ solubilization [PZ•] in which the
`denaturant and reducing agent are added to the broth at
`the end of the fermentatio\l process, and the cell debris is
`separated from the soluble material by aqueous two-phase
`extraction.
`
`Solubilized inclusion body proteins can be contaminated
`with varying levels of host proteins, nucleic acids, and
`the
`that
`thought
`is
`It
`cell membrane components.
`presence of these microbial contaminants may induce
`aggregation during refolding, thus reducing overall yields.
`Maachupalli-Reddy et al. [9°] showed that whereas non(cid:173)
`proteinaceous contaminants have little effect on renat(cid:173)
`uration yields, aggregation of protein contaminants can
`result in significant losses by triggering co-aggregation of
`the desired protein. Thus, some inclusion body processes
`include a purification step prior to refolding. Typically this
`step may be ion exchange [4,101, size exclusion [P3°,ll],
`metal affinity [12], or reverse phase chromatography [P3°].
`A common feature of these chromatographic steps is that
`they all operate with buffers that keep the protein in the
`denatured reduced state. If the solubilized protein is to
`be stored for later use, it may typically be exchanged
`into an acidic buffer, such as 10% acetic acid or 5-10 mM
`HCl [P3°,13] and freeze-dried. Exposure to low pH may
`result, for some proteins, in the formation of partially
`the native
`to
`to refold
`intermediates unable
`folded
`active configuration [14]. [n this case, the lyophilized
`protein should be resolubilized using chaotropic agents or
`detergents, before refolding is attempted.
`
`Renaturation of the solubilized protein
`Several methods, including dilution, dialysis, diafiltration,
`gel filtration, and immobilization onto a solid support, may
`be employed to remove or reduce excess denaturing and
`reducing agents, allowing proteins to renature. Dilution
`of the denatured solution directly into renaturation buffer
`is the easiest process. In dialysis, the denatured protein
`solution is dialyzed against renaturation buffer. Because
`dialysis is based on the diffusion of smaller molecules
`and ions through membranes, it may be too slow to
`be used in commercial scale production of proteins.
`intermediate
`In addition, exposure of the protein to
`concentration of denaturants for a prolonged period of time
`may cause aggregation. Diafiltration is a faster, therefore,
`
`more practical membrane-based alternative because the
`rate of denaturant removal is not diffusion limited, the
`driving force being pressure difference; however, as the
`driving force for buffer exchange is the pressure drop
`across the membrane, accumulation of denatured protein
`to
`limit its application due
`the membrane may
`on
`excessive fouling. Gel filtration chromatography has been
`successfully used to renature secretory leukocyte protease
`inhibitor, carbonic anhydrase and lysozyme [P4,15-17];
`however, problems in flow through the column may arise
`due tu protein aggregation upon buffer exchange. Aggre(cid:173)
`gation in a chromatographic column can be prevented
`by immobilizing individual polypeptide chains onto the
`matrix [12,13,18]. Potential complications may arise if
`folding of the protein is inhibited by binding to the
`solid support, which could be prevented by using fusion
`proteins [ 19,20°]. In addition to buffer exchange, column
`chromatography allows for some degree of purification of
`the desired product.
`
`In the case of disulphide bonded proteins, renaturation
`buffers must promote disulphide bond formation (oxi(cid:173)
`dation). The most common methods used to promote
`oxidation during refolding are: air oxidation; the oxido
`shuffling system; the use of mixed disulphides; and oxi(cid:173)
`dation of sulphonated proteins. Although, oxidation with
`air or oxygen in the presence of trace amounts of metal
`ions is simple and inexpensive [P2°,2 ! •], renaturation rates
`and yields can be low. Higher oxidation rates and yields
`can be obtained by utilizing 'oxido shuffling' reagents,
`low molecular weight thiols in reduced and oxidized
`forms, which allow for both formation and reshuffling
`of disulphide bonds, which can alter configurations. The
`most common oxido shuffling reagents are reduced and
`oxidized glutathione (GSH/GSSG), but the pairs cys(cid:173)
`teine/cystine, cysteamine/cystamine, OTT/oxidized glu(cid:173)
`tathione, and dithioerythritol/oxidized glutathione have
`also been utilized. Typically a 1-3 mM reduced thiol and
`a 10: 1 to 5: 1 ratio of reduced to oxidized thiol are used to
`promote proper disulphide bonding [21•]. More recently,
`we have shown that optimum renaturation yields are
`obtained when the ratio of reduced to oxidized thiol is
`anywhere between 3: 1 and I: I [22°]. A disadvantage of
`the oxido shuffling system over the use of air oxidation, is
`the high cost of some of the reagents, particularly oxidized
`glutathione.
`
`Another strategy employed to oxidize proteins during
`folding is the formation of mixed disulphides between
`oxidized glutathione and reduced protein before renat(cid:173)
`uration [3°]. Formation of mixed disulphides increases
`the solubility of the denatured protein by increasing the
`hydrophilic character of the polypeptide chain. Disulphide
`is then promoted by adding catalytic
`bond formation
`amounts of a reducing agent in the renaturation step. A
`similar protection of thiol groups during solubilization can
`be achieved by sulphonation of the denatured protein,
`in which a reducing agent and sodium sulphite are used
`
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`

`Refolding of recombinant proteins De Bernardez Clark
`
`159
`
`to cleave disulphide bonds and protect the resulting
`thiol groups as sulphonates [P3•,S•]. Under renaturation
`conditions, the protection groups are removed by oxidation
`in the presence of small amounts of a reducing agent to
`promote disulphide bond reshuffling.
`
`Competition between folding and aggregation
`incorrectly
`Formation of off-pathway species, such as
`folded species and aggregates, are the cause of decreased
`renaturation yields. Because aggregation is an intermolecu(cid:173)
`lar phenomenon, it is highly protein concentration depen(cid:173)
`dent. The most direct means of minimizing aggregation is
`by decreasing protein concentration. It has been suggested
`that optimum recovery yields can be expected if the
`protein concentration is in the range of 10-50 µg/ml [21 •].
`Renaturation at such low protein concentrations requires
`large volumes of refolding buffer, driving production
`costs upward.
`
`The key to a successful commercial refolding process
`lies in achieving high yields while refolding at high
`protein concentrations. One solution involves using either
`slow continuous or discontinuous addition of denatured
`protein to refolding buffer [3•]. Enough time is allowed
`between additions for the protein to fold past the early
`stages in the folding pathway, when it is susceptible to
`aggregation. The components of the solution containing
`the denatured protein must be carefully examined tu
`avoid detrimental effects due to their accumulation in the
`refolding solution after multiple addition steps. Another
`alternative for decreasing protein aggregation while folding
`at relatively high protein concentrations (up tu 4 mg/ml
`for carbonic anhydrase II) is to use the temperature-leap
`tactic [23], in which the protein is allowed to refold at
`low temperatures, to minimize aggregation, and then the
`temperature is rapidly raised to promote fast folding after
`the intermediates responsible for aggregation have been
`depleted. A third method involves folding by dilution tu
`final denaturant concentrations that are high enough to
`solubilize aggregates but low enough to promote proper
`folding. We have shown that the oxidative renaturation
`of lysozyme can be carried out at protein concentrations
`of up to 5 mg/ml with very high yields in the presence
`of 1-2 !vi GdmCl [22•]. An alternative method which also
`exposes the refolding protein to intermediate denaturant
`concentrations was developed by Maeda et al. [24 ]. In this
`method, renaturation is started by dialysis against a buffer
`containing high denaturant concentration (8 !vi urea) and
`thiol/disulphide exchange reagents, and the denaturant
`concentration in the dialysis buffer is gradually diluted
`using buffer without denaturant. Using this method,
`Maeda er al. [24] were able to refold immunoglobulin G
`at concentrations above 1 mg/ml with yields as high as
`70%. For proteins that do not tend to aggregate at
`intermediate denaturant concentrations, the slow dialysis
`method can successfully prevent aggregation by exposing
`the protein to a slow decrease in denaturant concentration.
`For proteins that aggregate at intermediate denaturant
`
`concentrations, fast or slow dilution of denatured protein
`into renaturation buffer, rather than slow dialysis, is the
`refolding method of choice.
`
`As aggregation is the major cause behind low renaturation
`yields, elucidating the aggregation pathway may hold the
`key to successful protein refolding at moderate to high
`protein concentrations. Intermediates with hydrophobic
`patches exposed to the solvent play a crucial role in the
`partition between native and aggregated conformations.
`Folding intermediates are believed to possess significant
`elements of secondary structure but little of the native
`tertiary structure. Due to the expanded volume of these
`intermediates, hydrophobic patches, which may normally
`be buried in the native state, are exposed to the solvent.
`When hydrophobic regions on separate polypeptide chains
`interact, intermediates are diverted off the correct folding
`pathway into aggregates. The so called 'molten globule'
`intermediate is believed to play a major role in the
`kinetics of folding [25] and probably plays a role in
`aggregation. Despite the controversy over the nature of
`this intermediate (on-pathway versus off-pathway) [26•]
`from a kinetic point of view, intermolecular association
`of molten globule-like intermediates may be the starting
`point of the aggregation pathway. On the other hand, Yon
`[27] suggests that intermolecular associations responsible
`for aggregate formation may arise from fluctuating species
`that precede the molten-globule state.
`
`Pioneer work by Goldberg et al. [28] shed light into
`the nature of interactions responsible for aggregation
`during folding. They showed that incorrect disulphide
`bonding may not he the major cause of aggregation
`because aggregates were formed even when a car(cid:173)
`boxymethylated protein was folded, that is, all cysteines
`are blocked from forming disulphide bonds. They also
`showed that aggregation is a non-specific phenomenon. On
`the other hand, Speed et al. [29•] recently reported that
`in mixed folding experiments using the P22 tailspike and
`coat proteins, folding intermediates of the two proteins
`did not coaggregate, but rather that they preferred to
`self-associate, suggesting that aggregation is a specific
`phenomenon. Since they only analyzed soluble aggregates,
`Speed et al. [29•] suggest that it is possible that larger
`aggregates could grow by a different mechanism involving
`nun-specific interactions.
`
`More recently, Maachupalli-Reddy et al. [9•] provided new
`evidence of the non-specific nature of the aggregation
`reaction by conducting mixed oxidative renaturation
`studies with hen egg-white lysozyme and three other
`proteins: 13-galactosidase, bovine serum albumin, and
`ribonuclease A. They found that foreign proteins that
`have a tendency to aggregate when folded in isolation,
`such as 13-galactosidase and bovine serum albumin,
`significantly decreased lysozyme renaturation yields by
`in mixed folding experiments.
`promoting aggregation
`On the other hand, ribonuclease A, which does not
`
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`

`160 Biochemical engineering
`
`significantly aggregate upon folding in isolation, did not
`in mixed folding
`affect lysozyme renaturation yields
`experiments. We have recently conducted experiments
`trying to understand the role chat disulphide bonding
`plays in the aggregation pathway [30]. We found that
`aggregation is fast and that aggregate concentration does
`the first minute of
`not significantly increase beyond
`renaturation. Hydrophobic interactions, and not disulphide
`bonding, were found to he the major cause of aggregation.
`Under renaturation conditions that promote disulphide
`bonding, however, aggregate size, but not concentration,
`was found to increase due to disulphide bond formation,
`resulting in covalently bonded aggregates. Based on
`these results, it is possible to speculate chat in mixed
`folding experiments, in which two or more proteins are
`simultaneously refolded, small soluble aggregates may
`form due to specific interactions that are hydrophobic in
`nature, and large heterogeneous aggregates may grow via
`disulphide bonding of unpaired cysteines, thus reconciling
`the conflicting observations of Speed et al. [29•] and
`Maaehupalli-Reddy et al. [9•] on the specific/non-specific
`nature of aggregates.
`
`An examination of aggregation data for the P22 tailspike
`protein, combined with the postulation of three possible
`mathematical models to describe the aggregation process,
`led Speed et al. [31] to conclude that aggregates grow
`via a cluster-cluster mulcimerization mechanism in which
`multimers of any size associate to form a larger aggregate.
`Aggregation is not mediated by the sequential addition
`of monomeric subunits and does not stop when the
`concentration of monomeric subunits is depleted. This
`confirms the observation (30] that aggregation is fast,
`total aggregate
`rather than
`that aggregate size,
`and
`concentration, increases as time progresses.
`
`Based on the hypothesis that aggregation is caused by
`interactions between hydrophobic patches in partially
`to envision
`folded polypeptide chains, it is possible
`to decrease aggregate formation. A careful
`strategies
`examination of structural and amino acid sequence data
`can lead to the identification of hydrophobic patches
`within the protein molecule that could participate in inter(cid:173)
`molecular interactions. Mutations causing the disruption of
`such hydrophobic patches may reduce aggregation. This
`strategy was tested by Pliickthun and co-workers [32,33•]
`who identified mutations located in turns of the protein
`and in hydrophobic patches which led to decreased in
`vitro and in vivo aggregation of recombinant antibody
`fragments. A second strategy involves the use of antibodies
`which preferentially bind hydrophobic patches away from
`the active site to protect the protein from intermolecular
`associations leading to aggregation. This strategy was
`tested by Katzav-Gozansky et al. [34•] who showed that
`carboxypeptidase A aggregation can be prevented using
`specific monoclonal antibodies. Interestingly, Pliickthun's
`group [32,33°] mutated amino acids likely to be on the sur(cid:173)
`face of the native protein, while Solomon and co-workers
`
`[34•] raised their antibodies using native antigents. These
`results seem to indicate that intermediates responsible for
`aggregation may have more native-like structural features
`than currently speculated.
`
`Improving renaturation yields
`A simpler strategy co prevent aggregation by interfering
`with intermolecular hydrophobic interactions is to use
`additives, small molecules that are relatively inexpensive
`and easy to remove once refolding goes to completion.
`A variety of additives have been tested for their ability
`to prevent aggregation. They may act by stabilizing the
`native state, by preferentially destabilizing incorrectly
`folded molecules, by increasing the solubility of folding
`intermediates, or by increasing the solubility of the
`unfolded state. In general, these additives do not seem
`to accelerate the rate of folding, but they do inhibit
`the unwanted aggregation reaction. Additives that have
`been shown to promote higher refolding yields are listed
`in Table 1.
`
`As Table 1 indicates, surfactants and detergents have
`proven to be very efficient folding aids, and have been
`shown to work with a variety of proteins, in particular
`multimeric disulphide bonded ones. Correct disulphide
`bond formation by thiol/disulphide exchange using oxido
`shuffling systems and air oxidation have been shown to
`be promoted in the presence of detergents (7,8•,35•].
`One drawback of the use of surfactants and detergents
`is that they are difficult to remove, a direct result of
`their ability to bind to proteins and to form micelles.
`Much easier to remove, low denaturant concentrations
`and L-arginine have shown excellent folding enhancing
`capabilities (Table 1); however, because they are used in
`the molar concentration range, they may interfere with the
`assembly of oligomeric proteins.
`
`As in vivo folding and aggregation processes are modulated
`by the presence of chaperones and foldases in the cellular
`environment, it is not surprising that such proteins can also
`impact the competition between folding and aggregation
`in in vitro protein folding [36•]. Chaperones and foldases,
`however, are proteins that need to be removed from the
`renaturation solution at the end of the refolding process,
`and may be costly to produce unless a recovery-reuse
`scheme can be implemented (37]. A practical solution
`implemented by
`this problem was proposed and
`to
`Altamirano et al. (38•] who used immobilized mini-chap(cid:173)
`to promote proper folding of several proteins
`erones
`which proved difficult to refold by other means. The
`immobilized mini-chaperones consisted of fragments of
`GroEL attached to chromatographic resins. The technique
`is only applicable to GroEL substrates and has not been
`tested under oxidative renaturation conditions.
`
`In an attempt to mimic the GroEL-GroES chaperonin
`action, Rozema and Gellman (35°,39] developed a folding
`strategy in which the denatured protein is first exposed
`
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`
`

`

`Table 1
`
`In vitro folding aids.
`
`Additive
`
`Non-denaturing concentrations of
`chaotropic agents
`GdmCI
`
`Urea
`
`L-arginine
`
`Salts
`Ammonium sulphate
`
`Sugars
`Glycerol
`
`Sucrose
`Glucose
`N-acetyl glucosamine
`
`Sarcosine
`
`Detergents and surfactants
`Chaps
`
`Tween
`SDS
`Sarkosyl
`Sodium lauorsylsarcosine
`Dodecyl maltoside
`Triton X-100
`Polyethylene glycol
`Octaethylene glycol
`monolauryl
`Phospholipids
`
`Sulphobetaines
`
`Shor1 chain alcohols
`n-pentanol
`n-hexanol
`cyclohexanol
`
`Protein
`
`Reference
`
`P. fluorescens lipase
`Hen egg-white lysozyme
`Carbonic anhydrase II
`lnterferon-~·polypeptides
`Porcine growth hormone
`Hen egg-white lysozyme
`IGF-1
`lnterferon-~·polypeptides
`
`P. fluorescens lipase
`Fab fragments
`Hen egg-white lysozyme
`a-gluocosidase
`
`1101
`(22•]
`(41]
`IP1I
`14]
`142"(
`(P2•I
`(P1J
`
`(1 DI
`(14)
`122•1
`120•1
`
`Hen egg-white lysozyme
`
`142·1
`
`P. fluorescens lipase
`Hen egg-white lysozymo
`IGF-1
`IGF-1
`Hen egg-white lysozyme
`Hen egg-white lysozyme
`
`Hen egg-white lysozyme
`
`TGF-P-like proteins
`Carbonic anhydrase II
`Human growth hormone
`lnterferon-p-polypeplides
`RNA polymerase q factor
`Single chain Fv fragment
`Class II MHC
`Carbonic anhydrase II
`Carbonic anhydrase II
`Carbonic anhydrase II
`
`Hen egg-white lysozyme
`TGF-P-like proteins
`
`Hen egg-white lysozyme
`~-D-galactosidase
`
`Carbonic anhydrase II
`Carbonic anhydrase II
`Carbonic anhydrase II
`
`[10)
`142"]
`[P2•]
`[P2•]
`142"]
`(42•]
`
`(42•]
`
`[P3•]
`[41]
`[44]
`IP1I
`(6]
`(7]
`(B•]
`(41]
`(41]
`[41]
`
`19•]
`(P3•]
`
`[431
`[431
`
`[411
`[411
`141]
`
`to a detergent-containing solution to prevent aggregation,
`followed by stripping of the detergent with cyclodexcrin to
`promote folding. The technique has been named 'artificial
`chaperone-assisted refolding' and has been applied to
`the refolding of carbonic anhydrase B [39], and the
`oxidative renaturation of lysozyme [35•]. This procedure
`has also been shown to work in the refolding MM-creatine
`kinase [40].
`
`Conclusions
`Inclusion body protein refolding used to be considered
`a difficult task. A protocol chat worked for one protein
`did not work for others. Finding the right conditions to
`fold a given protein was a trial and error process in which
`existing methods were tried until a successful one was
`found. This was in pare due to our lack of understanding
`of the competition between folding and aggregation in
`in vitro protein folding. Despite chis lack of knowledge,
`
`Refolding of recombinant proteins De Bernardez Clark
`
`161
`
`many efficient refolding process have been developed
`in which aggregation is reduced hy the use of additives
`that interfere with intermolecular interactions responsible
`for aggregation. As more and more additives are added
`to the list, there is a pressing need to characterize the
`aggregation process at the molecular level in order to
`sc:lect the right additive. Advances in our understanding
`of the aggregation pathway combined with knowledge on
`the role that chaperones play in in vivo protein folding
`provide the tools that will allow us to develop efficient
`refolding processes. Among these developments, finding
`sites on the protein molecule that interact with molecular
`chaperones, and identifying protein regions involved in
`intermolecular interactions will provide a rational basis
`for finding specific mutations and designing small binding
`molecules chat prevent aggregation.
`
`References and recommended reading
`Papers of particular interest, published within the annual period of review,
`have been highlighted as:
`
`of special interest
`•
`•• of outstanding interest
`
`1.
`
`2.
`
`Clarke AR, Waltho JP: Protein folding pathways and
`intermediates. Curr Opin Biotechnol 1997, 8:400-405.
`
`Herman R: Protein Folding. Munich: European Patent Office;
`1993. [EPO Applied Technology Series vol 12.]
`
`3.
`•
`
`Rudolph R, Bohm G. Lilia H, Jaenicke R: Folding proteins.
`In Protein Function. A Practical Approach, edn 2. Edited by
`Creighton TE. New York: IRL Press; 1997:57-99.
`This chapter covers relevant topics and techniques in protein unfold·
`ing/folding. It contains protocols for cell lysis, isolation of inclusion bodies,
`and renaturation of solubilized proteins with special emphasis on the renat·
`uration of disulphide bonded proteins. It also covers monitoring of protein
`folding, kinetics and equilibrium considerations, folding and association of
`oligomeric proteins, the effects of ligands and the effect of auxiliary proteins
`assisting protein foldlng.
`
`4.
`
`Cardamone M, Puri NK, Brandon MR: Comparing the refolding
`and reoxidation of recombinant porcine growth hormone from
`a urea denatured state and from Escherichia coli inclusion
`bodies. Biochemistry 1995, 34:5773-5794.
`
`5.
`•
`
`Cowley DJ, Mackin RB: Expression, purification and
`characterization of recombinant human proinsulln. FEBS Lett
`1997, 402:124-13D.
`E. coli derived recombinant human prolnsulin produced in inclusion bodies
`was solubilized with 700/o formic acid and cleaved with cyanogen bromide.
`After solvent evaporation and freeze-drying, the lyophilized protein was dis(cid:173)
`solved in 7 M urea and subjected to oxidative sulphitolysis. The sulphonated
`material was purified by anion exchange. and refolded at pH 10.5 in the
`presence of air and ~-mercaptoethanol. The refolded proinsulin contained
`the correct disulphide bond pattern.
`Burgess RR: Purification of overproduced Escherichia coli RNA
`polymerase cr factors by solubilizing inclusion bodies and
`refolding from sarkosyl. Methods Enzymol 1996. 273:145-149.
`
`6.
`
`7.
`
`Kurucz I, Titus JA, Jost CA, Segal DM: Correct disulphide
`pairing and efficient refolding of detergent-solubilized single·
`chain Fv proteins from bacterial inclusion bodies. Mo/ lmmunol
`t 995, 12:1443·1452.
`
`8.
`•
`
`Stockel J, Doring K, Malotka J. Jahnig F. Dornmair K: Pathway of
`detergent-mediated and peptide ligand-mediated refolding of
`heterodlmer class II major histocompatibility complex (MHC)
`molecules. Eur J Biochem 1997, 248:684-691.
`An in depth discussion of the mechanism of detergent-mediated protein fold(cid:173)
`ing of a heterodimeric disulphide bonded protein with four domains. The con(cid:173)
`tributions of detergent headgroup and aliphatic tail to the stabilization of fold(cid:173)
`ing intermediates are dissected. Optimal detergent concentration decreases
`with increasing critical micelle concentration. Formation of secondary struc(cid:173)
`ture occurs ear1y in the folding pathway when the denaturing detergent SDS
`is replaced by a mild detergent. Tertiary structure formation and heterodimer
`
`5 of 7
`
`Fresenius Kabi
`Exhibit 1021
`
`

`

`162 Biochemical engineering
`
`association occurs later in the folding pathway concomitantly with disulphide
`bond formation.
`
`25.
`
`Ptitsyn OB, Bychkova VE, Uversky VN: Kinetic and equilibrium
`folding intermediates. Philos Trans R Soc Land B Biol Sci
`1995, 348:35-41.
`
`9.
`•
`
`Maachupalli·Reddy J, Kelley BD, De Bernardez Clark E: Effect of
`inclusion body contaminants on the oxidative renaturation of
`hen egg white lysozyme. Biotechnol Prag 1997, 13:144·150.
`This paper shows that non-proteinaceous contaminants, such as plasmid
`ONA, ribosomal RNA, and lipopolysaccharides, have little effect on protein
`renaturation. Phosphalipids improve folding yields by about 15%. Proteina(cid:173)
`ceous contaminants, on the other hand, can have a significant detrimental
`effect on folding yields by causing co-aggregation of the protein of inter(cid:173)
`est. The paper also shows that contaminants affect the overall rate of the
`aggregation reaction without affecting the folding rate.
`
`1 0.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`Ahn JH, Lee YP, Rhee JS: Investigation of refolding condition
`for Pseudomonas f/uorescens lipase by response surface
`methodology. J B,otechnol 1997, 54:151-160.
`Simmons T, Newhouse YM, Arnold KS, lnnerarity TL,
`Weisgraber KH: Human low density lipoprotein receptor
`fragment Successful refolding of a functionally active ligand·
`binding domain produced in Escherichia coli. J Biol Chem
`1997, 272:25531-25536.
`
`Negro A, Onisto M, Grassato L, Caenazzo C, Garbisa S:
`Recombinant human TIMP-3 from Es