Chaperone-assisted refolding of Escherichia coli
maltodextrin glucosidase
Subhankar Paul
1,2
, Shashikala Punam
1
and Tapan K. Chaudhuri
1
1 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, India
2 Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, India
The protein folding problem remains one of the key
unsolved mysteries in biology [1,2]. Despite huge
research exercises and much recent advancement, it is
still unclear exactly how a disordered polypeptide
chain spontaneously folds into a uniquely structured,
biologically active protein molecule [3–6].
In his work on the refolding of ribonuclease, Anfin-
sen [7] concluded that the unique tertiary structure of
a protein is determined by its amino acid sequence,
and that the protein recovers its complete native struc-
ture when the denaturing stress is withdrawn, indicat-
ing that the unfolding and refolding of proteins is a
reversible phenomenon. The general validity of this
conclusion was later proved to be wrong as a number
of proteins, such as subtilisin E [8], a-lytic protease [9]
and carboxypeptidase Y [10], failed to refold correctly
from the unfolded state. During refolding, many pro-
teins formed aggregates of misfolded proteins whereas
Keywords
chemical chaperone-assisted refolding;
GroEL; GroES; MalZ; protein aggregation
Correspondence
S. Paul, Department of Biotechnology and
Medical Engineering, National Institute of
Technology Rourkela, Rourkela 769008,
India
Fax: +91 661 2462999
Tel: +91 661 2462284
E-mail: subhankar_paul@rediffmail.com,
spaul@nitrkl.ac.in
T. K. Chaudhuri, Department of Biochemical
Engineering and Biotechnology, Indian
Institute of Technology Delhi, Hauz Khas,
New Delhi 110016, India
Fax: +91 11 2658 2282
Tel: +91 11 2659 1012
E-mail: tapan@dbeb.iitd.ac.in
(Received 4 August 2007, revised 27 Sep-
tember 2007, accepted 1 October 2007)
doi:10.1111/j.1742-4658.2007.06122.x
In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escher-
ichia coli protein, was studied in the presence of glycerol, dimethylsulfox-
ide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and
chaperonins GroEL and GroES. Different osmolytes, namely proline, glyc-
erol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemi-
cal chaperones, assist in protein folding through effective inhibition of the
aggregation process. In the present study, it was observed that a few chemi-
cal chaperones effectively reduced the aggregation process of maltodextrin
glucosidase and hence the in vitro refolding was substantially enhanced,
with ethylene glycol being the exception. Although, the highest recovery of
active maltodextrin glucosidase was achieved through the ATP-mediated
GroEL ⁄GroES-assisted refolding of denatured protein, the yield of cor-
rectly folded protein from glycerol- or proline-assisted spontaneous refold-
ing process was closer to the chaperonin-assisted refolding. It was also
observed that the combined application of chemical chaperones and mole-
cular chaperone was more productive than their individual contribution
towards the in vitro refolding of maltodextrin glucosidase. The chemical
chaperones, except ethylene glycol, were found to provide different degrees
of protection to maltodextrin glucosidase from thermal denaturation,
whereas proline caused the highest protection. The observations from the
present studies conclusively demonstrate that chemical or molecular chap-
erones, or the combination of both chaperones, could be used in the effi-
cient refolding of recombinant E. coli maltodextrin glucosidase, which
enhances the possibility of identifying or designing suitable small molecules
that can act as chemical chaperones in the efficient refolding of various
aggregate-prone proteins of commercial and medical importance.
Abbreviations
EG, ethylene glycol; GdnHCl, guanidine hydrochloride; MalZ, maltodextrin glucosidase; TMAO, trimethylamine-N-oxide.
6000 FEBS Journal 274 (2007) 6000–6010 ª2007 The Authors Journal compilation ª2007 FEBS
some refolded to a non-native conformation [11,12].
The conformation of the refolded protein depends not
only on the nature of the unfolding and refolding con-
ditions, but also on the molecular mass of the protein.
Low molecular mass proteins showed a tendency to
fold reversibly [13].
For comparatively high molecular weight proteins,
misfolding and aggregation is a commonly observed
phenomenon in their folding pathways [14–17]. Efficient
refolding of proteins and prevention of their aggrega-
tion during folding has a huge importance in recombi-
nant protein production and in finding cures for several
genetic disorders. Correct folding in vitro or in vivo
competes with unproductive side reactions such as mis-
folding or aggregation [18]. Aggregation of proteins
during folding, both in vitro and in vivo, is known to
lead to poor native protein yields as well as the onset of
several age-related diseases [19]. Hence, there is a grow-
ing interest in developing strategies to prevent protein
aggregation for enhancing protein–refolding yields and
designing new drugs countering many protein-misfold-
ing diseases. Several attempts have been made in this
direction with successes as well as failures [20–22].
In vitro, osmolytes, such as the small molecules beta-
ine, proline, trehalose, glycerol, dimethylsulfoxide,
trimethylamine-N-oxide (TMAO) and ethylene glycol
(EG), have been reported to protect native proteins
from heat denaturation and favor the formation of
native protein oligomers [23–29]. They serve as stabiliz-
ers of proteins and cell components against the dena-
turing effect of ionic strength. Furthermore, these
osmolytes behave as ‘chemical chaperones’ by promot-
ing the correct refolding of proteins in vitro and in the
cell and by protecting native proteins from heat dena-
turation. Some osmolytes behave as chemical chaper-
ones by promoting the correct folding of unfolded
protein in vitro and in vivo [26,27,30–32]; for example,
proline behaves as a protein folding chaperone [26].
In the present study, we investigated the effect of a
few osmolytes ⁄chemical chaperones, such as glycerol,
dimethylsulfoxide, TMAO, trehalose, EG and proline,
on the refolding of a Escherichia coli protein maltodex-
trin glucosidase (MalZ), a 69 kDa monomeric protein
responsible for the degradation of maltodextrins to
maltose by eliminating one glucose residue from the
reducing end at each time. We also aimed to compare
the efficiency of osmolyte-mediated refolding with the
most popular cellular chaperones, GroEL and GroES,
in the assisted refolding of MalZ in vitro. It has
recently been reported that, in the presence of ATP,
GroEL and GroES assist the folding of aggregation
prone recombinant MalZ in vivo and the same study
also demonstrated that, when MalZ was refolded from
a guanidine hydrochloride (GdnHCl) denatured state,
GroEL, GroES and ATP together recovered the MalZ
activity substantially [33] and significantly increased
the refolding yield of MalZ. The present study demon-
strates that all the osmolytes ⁄chemical chaperones,
with the exception of EG, enhance the extent of refold-
ing of MalZ significantly over the spontaneous yield
and the recovery of folded MalZ in glycerol and pro-
line-assisted refolding was comparable to the GroEL ⁄
GroES-assisted recovery of MalZ. It is also demon-
strated that chemical chaperones ⁄osmolytes protected
MalZ from thermal denaturation, as well as denatur-
ation induced by chaotropic agents such as urea. It is
further observed that the combined application of
chemical chaperones and the molecular chaperones
GroEL ⁄GroES yielded a higher recovery of refolded
MalZ than the yield obtained through their individual
assistance.
Results
Deactivation of MalZ by urea
Denaturation of proteins using high concentrations of
chaotropic agents (e.g. GdnHCl or urea) is a well
known technique that is normally used to study the
unfolding of proteins. Such chemically induced dena-
turation of proteins results in a gradual loss of their
secondary and tertiary structures. In the present study,
8murea and 20 mmdithiothreitol in 20 mmsodium
phosphate buffer solution pH 7.0 was used to denature
MalZ to ensure the complete denaturation of the
enzyme. Deanturation of MalZ was monitored by the
complete loss of MalZ activity (Fig. 1A) and the loss
of relative tryptophan fluorescence intensity (Fig. 1B)
with increasing concentration of urea.
Effect of protein concentration on refolding yield
For in vitro refolding of MalZ, correct folding com-
petes kinetically with misfolding as well as aggregation.
Unproductive aggregation may primarily originate
from hydrophobic interactions of unfolded polypeptide
chains as second or higher order processes. Aggrega-
tion of MalZ is concentration dependent as observed
from the experimental results (Fig. 2). It has been
reported previously that the refolding yield of many
proteins depends on the protein concentration [34,35].
It was observed that there was a drastic reduction in
the spontaneous refolding yield of MalZ with increased
concentration of the protein, with negligible refolding
observed beyond the MalZ concentration of
50 lgÆmL
)1
(Fig. 2).
S. Paul et al.Chaperone-assisted folding of maltodextrin glucosidase
FEBS Journal 274 (2007) 6000–6010 ª2007 The Authors Journal compilation ª2007 FEBS 6001
Folding of MalZ
When urea denatured MalZ was diluted into 20 mm
sodium phosphate buffer, pH 7.0, the recovery of
refolded protein was almost negligible; however, the
extent of refolding was enhanced in the presence of
reducing agent dithiothreitol in the dilution buffer at a
specific concentration. Subsequently, the yield of
refolded protein was found to be significantly increased
by the addition of MgCl
2
in the refolding buffer. The
optimal concentration of the reductant dithiothreitol
was found to be 5 mm(Fig. 3B) and that of the Mg
+2
ion was 1 mm(Fig. 3A). Dilution of the denatured
MalZ into the renaturing buffer containing these two
inorganic cofactors restored approximately 10% of
the activity of the recombinant enzyme maltodextrin
glucosidase.
Table 1 shows the effect of concentration of various
osmolytes (i.e. glycerol, dimethylsulfoxide, TMAO, tre-
halose, EG and proline in the concentration range
1–9 m), depending on their solubility, on the refolding
yield of MalZ at 30 C. All osmolytes led to an initial
increase in the refolding yield, followed by a decrease
at higher concentrations, but EG, which is known as a
destabilizer of protein conformation, showed almost
no improvement in the refolding yield of MalZ over
the spontaneous yield, with a maximum refolding yield
of 12% at 2 mconcentration. For dimethylsulfoxide,
there was a sharp decline in the yield of refolding
beyond 3 m, with very little refolding being obtained
at 8 m(Table 1). Glycerol, which has long been known
to stabilize the native structure of proteins against
chemical and thermal denaturation, also led to a grad-
ual increase in the refolding yield, and a very high
refolding yield of 61% was obtained at 2 m, followed
by a decrease of the yield. It was not possible to use
glycerol at concentrations higher than 8 mdue to high
viscosity of the solutions.
Effect of temperature on the refolding yield
To observe the effect of temperature on the refolding
yield in the chemical as well as molecular chaperone-
0
20
40
60
80
100
A
B
[Urea]
0
100
200
300
400
500
600
700
[Urea]
02468
02468
Relative fluotrescence intensity of MalZ Relative MalZ activity
Fig. 1. Unfolding of MalZ (5 lM) was carried out by the addition of
urea at pH 7.0 in 20 mMsodium phosphate buffer containing
20 mMdithiothreitol. (A) MalZ samples were incubated for 4 h at
30 C with different concentrations of urea in the range 0–8 M.
MalZ enzymatic activity with increasing concentration of urea was
measured and plotted against the concentration of urea. Percent-
age of residual activity was expressed relative to activity obtained
from same amount of native protein. (B) MalZ samples were incu-
bated for 4 h at 30 Cin8Murea and in 20 mMdithiothreitol. Dif-
ferent concentrations of urea were used for equilibrium unfolding
of MalZ. Relative intrinsic fluorescence emission was measured at
346 nm for all the samples at the kEx
max 279 nm. The excitation and
emission band pass were 5 nm and 7.5 nm, respectively, and the
scan rate was 60 nmÆmin
)1
.
0 102030405060
0
5
10
15
20
25
30
% Relative activity of MalZ
MalZ conc. µg/µL
Fig. 2. Effect of protein concentration on the spontaneous refolding
yield of MalZ at 30 C.
Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al.
6002 FEBS Journal 274 (2007) 6000–6010 ª2007 The Authors Journal compilation ª2007 FEBS
assisted in vitro refolding of MalZ, the protein was
denatured by 8 murea and incubated for 4 h at room
temperature. Refolding reactions were carried out at
various temperatures (10, 15, 20, 25, 30, 35 and 40 C)
by diluting the denatured protein solution with 20 mm
sodium phosphate buffer, pH 7.0, containing respective
chemical chaperones or GroEL ⁄GroES ⁄ATP. The
spontaneous refolding was measured to be approxi-
mately 13% at 25 C (Table 2). Contrary to spontane-
ous refolding, chemical chaperones and E. coli
molecular chaperones GroEL ⁄GroES increased the
refolding yield of MalZ to a better extent. Glycerol,
proline and GroEL ⁄GroES particularly increased the
recovery of active MalZ significantly at higher tempera-
ture. Although GroEL ⁄GroES-mediated recovery of
folded MalZ at a temperature of 40 C was found to be
highest (approximately 66%), the yield of folded MalZ,
assisted by glycerol and proline, was also close to the
recovery obtained by GroELS-assisted refolding
(Table 2). Although the spontaneous refolding of MalZ
was maximum at 20 C (approximately 16%), this yield
was much less than the chemical chaperones or
GroEL ⁄GroES assisted refolding yield under the same
conditions. The spontaneous refolding yield was
reduced drastically with increasing temperature and, at
40 C, it was only 3%.
Osmolyte-induced protection of MalZ activity
in vitro
To examine how various cosolvents protect the native
structure of MalZ, an increasing concentration of urea,
in the range 0–8 m, was used to denature MalZ and
the denaturation was monitored by measuring the loss
of biological activity of MalZ. Optimized concentration
of different chemical chaperones in the denaturation
mixture and, in every case, their effect on protecting
the biological activity of the protein was monitored.
The control refolding experiment of MalZ was car-
ried out in absence of any osmolyte. It was observed
Table 1. The effect of different concentrations (M) of chemical chaperones on the refolding yield of MalZ at 30 C. The data are an average
of at least three independent observations with a maximum percentage error of ± 5%.
Chaperone
concentration (M)
Refolding yield of MalZ (%) in presence of following chemical chaperones
Glycerol Dimethylsulfoxide TMAO Trehalose EG Proline
0111111111111
1362924421258
2614530331131
3423549221128
4323240211026
525283816921
6152122–518
7111518–517
86118––9
9––7––6
0
2
4
6
8
10
12
14
A
B
[MgCl
2
] (m
M
)
0
2
4
6
8
10
12
14
0246810
% Refolding yield of MalZ
% Refolding yields of MalZ
[dithiothreitol] (mM)
0 2 4 6 8 10 12 14 16
Fig. 3. Determination of the optimum concentrations of dithiothrei-
tol and MgCl
2
for the in vitro refolding of MalZ. (A) The denatured
solution of MalZ in 8 Murea was diluted 100-fold into 20 mM
sodium phosphate, pH 7.0, containing various concentrations of
MgCl
2
. (B) The renaturation was carried out with 1 mMMgCl
2
and
various concentrations of dithiothreitol. The enzyme solutions were
incubated at room temperature for 4 h, and the activity assay was
performed as described by Tapio et al. [41].
S. Paul et al.Chaperone-assisted folding of maltodextrin glucosidase
FEBS Journal 274 (2007) 6000–6010 ª2007 The Authors Journal compilation ª2007 FEBS 6003
that all the polyols protected the MalZ from denatur-
ation and deactivation to certain extent (Fig. 4). Pro-
line and glycerol, among all the osmolytes, exhibited
the highest degree of protection to the MalZ activity.
For example, at 4 murea, when the activity of MalZ is
almost negligible, glycerol and proline helped the pro-
tein to retain approximately 40% of its initial activity.
Although dimethylsulfoxide, TMAO, trehalose and
EG have lower protection ability than glycerol and
proline, they provided a fair level of protection
towards the MalZ activity.
Chemical chaperones enhance the refolding yield
of MalZ in vitro
MalZ was denatured by 8 murea and dithiothreitol
and incubated at 30 C for 4 h. Urea denatured MalZ
was refolded by 100-fold dilution with refolding buffer
(20 mmsodium phosphate buffer, pH 7.0, containing
5mmdithiothreitol and 1 mmMgCl
2
). Chemical chap-
erone-mediated refolding was carried out by diluting
denatured MalZ with the refolding buffer containing
the desired concentration of various chemical chaper-
ones. To monitor spontaneous refolding, denatured
MalZ (10 lm) was diluted directly into refolding buffer
(20 mmsodium phosphate, pH 7.0, containing 5 mm
dithiothreitol and 1 mmMgCl
2
). Molecular chaperone-
assisted refolding was carried out by diluting the dena-
tured MalZ with the refolding buffer containing
GroEL (in such a manner that the final concentration
of MalZ and GroEL was 0.1 lmin the solution). After
10 min of incubation at 30 C, ATP (5 mmfinal con-
centration) and GroES (0.2 lmfinal concentration)
were added to the refolding mixture. The negative con-
trol for all these experiments comprise of the buffer
containing 0.08 murea, which corresponds to the
residual concentration of urea in the refolding mixture,
and the positive control was the buffer containing
0.1 lmnative MalZ protein and 0.08 murea. Refold-
ing mixtures were withdrawn at different time intervals
and MalZ activity was assayed for different samples at
different time intervals up to 10 h. The percentage
recovery of MalZ activity in different samples was
calculated after considering the equivalent amount of
native MalZ activity as 100%.
The spontaneous refolding of MalZ was approxi-
mately 11% (Table 3). When the refolding reaction
was carried out in the presence of various chemical
chaperones, a significant enhancement of refolding
yield was observed over the spontaneous refolding
yield, with EG being the exception. For all cases, there
was a gradual increase of refolding yield with time
and, after 5 h of refolding, no increase in yield was
observed. The use of glycerol and proline resulted in
the most significant improvement of refolding recovery
over the spontaneous one (61% and 58% refolding
yield, respectively). However, the common trend for
02468
0
20
40
60
80
100
[urea] (M)
% Relative MalZ activity
Fig. 4. Deactivation of MalZ (5 lM) by increasing concentration of
urea in the presence of an optimized concentration of TMAO (.),
glycerol (m), dimethylsulfoxide (d), trehalose (r), EG (+) or proline
(·), or in absence of any osmolytes (j), at 30 C.
Table 2. Percent of refolding compared to native MalZ, which was treated in the same way as the denatured protein at the respective tem-
peratures.
Temperature
(C) Spontaneous
The effect of different concentrations (M) of chemical chaperones on the percentage refolding yield of
MalZ at 30 C
Dimethylsulfoxide Glycerol TMAO Trehalose EG Proline GroEL ⁄GroES
10 09 17 22 18 15 07 20 21
15 15 21 25 23 21 10 26 35
20 16 28 37 29 25 11 31 44
25 13 42 44 44 35 12 47 52
30 11 45 61 49 42 14 58 55
35 9 51 65 50 42 15 68 58
40 3 51 62 50 40 12 64 66
Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al.
6004 FEBS Journal 274 (2007) 6000–6010 ª2007 The Authors Journal compilation ª2007 FEBS