497
Advances in refolding of proteins produced in E. coli
Hauke Lilie, Elisabeth Schwarz and Rainer Rudolph*
Inclusion body production is a common theme in 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
procedures include the inhibition of aggregation during
refolding by the application of low molecular weight additives
and matrix-bound renaturation techniques.
Addresses
Institutfor Biotechnologie,Martin LutherUniversitAtHalle-Wittenberg,
Kurt Mothes Strasse 3, D-06120 Halle, Germany
*e-mail: rudolph@biochemtech.uni-halle.de
Current Opinion in Biotechnology 1998, 9:497-501
http://biomednet.com/elecref/0958166900900497
~e,Current BiologyLtd ISSN 0958-1669
Abbreviation
GdmCl guanidiniumhydrochloride
Introduction
Elegant and well established recombinant D N A method-
ologics have set the stage for the production of
heterologous proteins in microbial hosts. T h e abundance
of protein expression systems renders the efficicnt bacter-
ial production of most proteins possible; howcvct, high
level expression of recombinant protein often results in
aggregation and accumulation in inchision bodies.
l)epositinn of the recombinant protcin in inchlsion bodics
can be hcaxcn or hell. T h e latter applies to those proteins
for which rcnaturation is problematical. Here, the only way
out is the awfidance or at least reduction of inclusion body
tk~rmation. In contrast, in the case of a simple and efficient
renaturation procedure, deposition of the protein in inclu-
sion bodies and subsequent isolation and renaturation of
inclusion bodv protein often means the most straightfof
ward strategy to get large amounts of the recombinant
protein. In this revicw we present an extract of recent
developments in inchision body refnlding.
Inclusion body formation
lTpon overexpressinn of recombinant proteins, inclusion
bodies can be observed in several host systems, for exam-
pie, prokaryotes, yeast or higher eukaryotcs. Even
endogenous proteins, if overexpressed, can accumulate in
inclusion bndies [1], snggcsting that in most cases inclu-
sion body formation is a conseqnence of high expression
rates, regardless of the svstem or protein used. T h e r e is no
direct correlation bctween the propensity of inchision
body formation of a certain protein and its intrinsic prop-
erties, such as molecular weight, hydrophobicity, folding
pathways, and so on. Only in the case of distllfide bonded
proteins can inclusion body formation be anticipated if the
protein is produced in the bacterial cytosol, as formation of
distllfide bonds does usually not occur in this reducing cel-
lular compartment. T h e consequencc is improper folding
resulting in aggregation.
An increase in the concentration of non-native polypep-
tides due to high cxpression levels seems to be responsible
for aggrcgation of the recombinant protein. This assuntp-
tion was quantified in a kinetic model that analysed the
yield of native protein as a flmction of the competition
between folding and aggregation [2]. According to this
model, the rclative yield of natixe prntcin increased with a
dccrcased rate of protcin synthesis. Qualitativcl> this was
confirmed by recombinant protcin expression at optimal
and suboptintal conditions. Thus, whereas recombinant
protcins often aggregate when b;.~c~eri~ia coli cells are cul-
tivated at 37°(], reduction of the cultivation temperatLlre
can increase the amount of natixe protein due to a
decrease of the rate of protein synthesis [3].
Alternatively, the addition of non-metabolizable carl)on
sources, such as desoxyglucnse, at the time of induction
leads to a reduced metabolic rate, which results in a limit-
ed production of the recombinant protcin. Another
possibility is limited induction of gene expression by thc
promotors, which can bc linearily regulated bv the induc-
er concentration. Recombinant protein deposition in
inchlsion bodies is commonly observed with hydrophobic
proteins. Here, fusion with a hydrophilic protein can
enhance sohibility. \Videlv uscd fusion parmer proteins are
glutathionc-S-transferase, maltose-binding protein or
thiorednxin [4,5].
A way of taking advantage of thc host cell's cquipment to
deal with protein folding is the co-overexpression of the
rccombinant protein with molecular chapcr()nes [6,7",8,9].
Still, the beneficial effect of co-overexprcssion of chaper-
one proteins is unpredictable, :is the appropriate
substratc-chaperone combination is a matter of trial and
error [10].
In the case of disulfide-containing proteins, the principles
mentioned above are not sufficient to aw)id inchision body
formation. Although the redticing conditions of the bacte-
rial cvtosol allow distllfide bond formation in a few cases
[11], distllfides are not normally tT)rmed in this compart-
ment. In contrast, the prokaryotic periplasm provides
oxidizing conditions for distllfide bonding; however, inclu-
sinn body formation can also occur in the periplasm [12].
In particular, the native periplasmic expression of proteins
that possess several distllfide-bonds is problematical as the
endogenous periplasmic enzyme distllfide bond oxidore-
ductase (DsbA) merely introduces distllfides into proteins,
but does not catalyze distllfide reshuffling. A means to
enhance correction of incorrect disulfide-bonds in the
498 Expressionvectors and delivery systems
Figure 1
E. coil expressing the heavy chain of the antibody MAK 33. The cells
were harvested six hours after induction of expression. Preparation for
electron microscopy included fixation with glutaraldehyde, embedding
in Epon resin and negative staining with uranyl acetate. The inclusion
bodies are visible as amorphous light grey structures.
periplasm of E. co/i is to overexpress the endogenous
periplasmic I)sbC protein, which is a disulfidc isomerase
[13", 14]. Also, cultivation in the presence of thinl reagents,
which lead to reshuffling of incorrect disulfide bridges, has
been proven to enhance the yield of native proteins con-
taining multiple disulfide bridges [15,Pl ].
Properties of inclusion bodies
Inclusion bodies are very dense particles of aggregated
protein (Figure 1). Because of their retractile property they
can be visualized by light microscopy. Inclusion bodies
may reach sizes with a diameter in the lumg range and
exhibit an amorphous or paracrystalline structure indepen-
dent of their subcellular location. Under appropriate
conditions the recombinant protein deposited in inclusion
bodies amounts 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 copuri|) ~ 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 properties of the
aggregated protein. Structural analyses of inclusion body
proteins indicate that the aggregates possess a certain
amount of secondary structure [16], a result also observed
for in vitro aggregated proteins [17].
Being aggregated polypeptides, inclusion bodies are gener-
ally not very sensitive to proteolytic breakdown. Still,
degradation products can often be detected in inclusion
bodies. After analysis of the expression kinetics, an
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 the
aggregation process further decreases protcolysis.
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 important that cell lvsis is
complete, because intact cells sediment together with the
inclusion bodies, thus, contaminating the preparation with
host protein. The most effective procedure for colnplete
cell disruption is a high pressure dispersion following a
lysozyme treatment. A fl, rther treatment of the crude
lysatc with detergents solubilizes lipids and membrane
proteins (fnr protocol see [19"]). In a few cases, urea or
guanidinium hvdrochloride ((;dmC1) at low concentrations
can dissociate inclusion body associated proteins. It shot, ld
be kept in mind, however, that a too high urea or (;dm('A
concentration will lead to the solubilization of the inclu-
sion bodies themselves. On average, an inchlsion body
preparation contains more than 50% of the recombinant
protein, the purity of the preparation may reach t,p to 90%
under optimal conditions.
Before starting renaturation the inclusion body material
has to be solubilized by strong denaturants, such as 6 M
GdmCl or 8 M urea. The addition of dithiothreitol keeps
all cysteines in the reduced state and cleaves disulfide
bonds formed during preparation. Before renaturation dial-
ysis against GdmCl at low pH without reducing agents can
be performed to remove the dithiothreitol for subsequent
storage (for protocol see [19"]).
R e n a t u r a t i o n of inclusion b o d y p r o t e i n s
Renaturation of solubilized inclusion bodies is initiated by
the removal of the denaturant either by dialysis or dilution.
As discussed for the in vivo situation, the efficiency of
renaturation depends on the competition between correct
folding and aggregation (Figure 2). Aggrcgation of the non-
native recombinant protein may be enhanced by other
proteins contaminating the inclusion body preparation 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 gg/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 may be relative-
ly low, necessitating large volumes for preparation of large
quantities of the native protein. Because during refolding
only the concentration of non-native polypeptides and not
that of native protein is critical for aggregation, a strategy
to circumvent the problem of large refolding vessels is
pulse renaturation [21]. In order to keep the concentration
of the unfolded protein low, thus limiting aggregation,
 Advances in refolding of proteins produced in E. coil Lilie, Schwarz and Rudolph
Figure 2
CLDH (pg/rnt)
1 10 10
100 k 100
g
50 50
#_
o
I
0 within tile peptide. Because tile peptide did not possess
001 oi 1 10
CLDH (HM)
Effect of protein concentration on the renaturation of lactate
dehydrogenase (taken from [18]). Acid denatured lactate
dehydrogenase was renatured in 0.1 M phosphate buffer pH 7, 1 mM
EDTA, 1 mM DTE, 20°C. After 192 hours reactivation (O) and
aggregation (A) were quantified.
aliquots of denatured protein are added at defined time
points to the renatt, ration buffer. T h e time interval
between two pulses has to bc optimized accnrding to the
refl)lding and aggregation behaviour of tile respective pro-
tein. T h e pulse renaturation is stopped x~hen the
concentration of denaturant, introduced in the refl)lding
buffer together with the denatured pn)tein, reaches a crit-
ical level, that is, a concentration at which even the native
protein develops the propensity to aggregate.
If proteins contain disulfide bonds, the renaturation buffer
has to be supplemented with a redox system. T h e addition
of a mixture of the reduced and oxidized forms of low mol-
ecular weight thiol reagents, such as glutathione, cysteine
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]. T h e redox system described
above is characterized by overall reducing conditions.
('a)rrect disulfides a r e protected bv the stable native struc-
ture; however, those corrcct disulfide bonds within the
native structure which are still soh'ent 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 copper-induced air oxidation can
ensure formation nf the respective disulfide bonds.
For certain proteins, probably due to low solubility of fold-
ing intermediates, oxidative refolding is not very effective.
In this case, the yield of renaturation may be increased if 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 to mixed disulfides.
Renaturation of the protein is pcrformed by dilution in a
renaturatinn buffer containing catalytic amounts of reduced
499
glutathione [P2]. T h e advantage of this procedure is that tile
mixed disulfide form of the protein carries additional
charged residues provided by the glutathione moiety, which
increases the solubility of the protein during refolding.
Recent studies on disulfide bond formation within pep-
tides showed the possibility of a directed reaction.
j Oxidation of peptides containing both two selenocysteines
and two cysteines lcads to the formation of two disulfides:
one between the selenocysteine residues and another
<~
between tile cysteines. No disufide bond between seleno-
ZS cvsteine and cysteine was observed [25]. This resuh was
independent of the order of selenocysteines and cvsteines
any conformational constraints directing disulfide bond
formation, these results clearly demonstrate the specifc
reactivity due to the different chemical properties of
selenocysteines and cysteines. Whether such disulfide
engineering can be transposed on proteins to prevent the
formation of wrong disulfides remains to be seen.
In addition to tile 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
renaturation [26,27]. Specific cofactors, sucb as Zn z+ or
Ca e+, can stabilize proteins already at the level of folding
intermediates, thus, preventing off-pathway reactions.
Besides such cofactnrs 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, a r e
essential for the renaturation of reduced chymotrypsino-
gen A and have been shown to promote fi)lding of several
other proteins [P3]. On the other hand, by slowing down
tile refi)lding kinetics, GdmCl and urea can shift the con>
petition between renaturation and aggregation towards the
aggregation 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 about 80% in the presence of 0.5 ]M L-arginine,
whereas in the absence of this additive almost no reactix i-
ty was observed [P2]. T h e mechanism by which L-arginine
supports renaturation is still unknown. I,-arginine slightly
destabilizes proteins [31] in a manner comparable to low
concentrations of chaotrophs. T h e b e n e f c a l cffect of
l,-arginine on refolding, however, probably originates from
an increased solubilization of folding intermediates.
Likewise, increased solubilization of folding intermediates
can explain the positive effect of detergents on the refolding
yield. Both ionic and non-ionic detergents can be used to
suppress aggregation upon dilution of the denatured protein
in the renaturation buffer. Using laurylmaltosid, Chaps (3-[3-
chloramidopropyl] dinaethylammonia-l-propane sulfonate)
or some other detergents during renaturation, the yield of
refolded protein can be improved [32-34,P4]. Other
 500 Expression vectors and delivery systems
detergents, inhibiting renaturation, have to be removed after
dilution by addition of cyclodextrin, which specifically binds
detergent molecules [35].
Another possibility for suppressing unspecific intermolecu-
lar interactions is the coupling of the denatured protein to a
matrix. Denatt,red 0¢-glucosidase fused to a poly-arginine
tag was bound to Heparin-Sephamse. Renaturation under
conditions at which the protein is still bound to the matrix
resulted in high yields of active protein even at protein con-
centrations in a mg/ml range [36]. Another matrix used for
this kind of renaturation was Ni-NTA agarose, originally
developed for an efficient protein purification. After binding
the denatured protein to the matrix via a His-tag the column
is equilibrated with renaturation buffer and the rcfolded
protein can be eluted by an imidazole or pH gradient. First
demonstrated for chloramphenicol acetyl transferase,
recently, the oxidative renaturation of a disulfide bridged
protein T1MP 3 on a Ni-agarose was described [37,38].
Conclusions
'lbdays detailed knowledge of the mechanisms of protein
f(4ding and its nff-pathwav reactions, as well as the interre-
lation of protein structure and folding, makes it possible to
basically design renaturation experiments. Still, the specif-
ic conditions regarding buffer composition, protein
concentration, temperature, and so on, has to be optimized
for every protein. Failure of renaturation may bc caused by
omission of cofactors, such as stnmturally important metal
ions, or degradation by traces of proteases. For those pro-
teins which are synthesized as proforms i,I vitro, the
prosequence may be crucial for structure formation and has
to be included in the refolding scheme [39]. Structural and
fimctional analyses of proteins, especially for therapeutic or
other industrial applications, require large amounts of pro-
teins. Inclusion body production and protein renaturation
prnvidcs an efficient route to meet these requirements.
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