Báo cáo khoa học: Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase doc

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ture when the denaturing stress is withdrawn, indicat-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: ,

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:
(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 ª 2007 The Authors Journal compilation ª 2007 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,
8 m urea and 20 mm dithiothreitol in 20 mm sodium
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 ª 2007 The Authors Journal compilation ª 2007 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 m concentration. 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 m due 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 m
M sodium phosphate buffer containing
20 m
M dithiothreitol. (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 °Cin8
M urea and in 20 mM dithiothreitol. 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 k
Ex
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 ª 2007 The Authors Journal compilation ª 2007 FEBS
assisted in vitro refolding of MalZ, the protein was
denatured by 8 m urea 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
M urea 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 mM MgCl
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 ª 2007 The Authors Journal compilation ª 2007 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 m urea, 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 m urea and dithiothreitol
and incubated at 30 °C for 4 h. Urea denatured MalZ
was refolded by 100-fold dilution with refolding buffer
(20 mm sodium phosphate buffer, pH 7.0, containing
5mm dithiothreitol and 1 mm MgCl
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 mm sodium phosphate, pH 7.0, containing 5 mm
dithiothreitol and 1 mm MgCl
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 lm in the solution). After
10 min of incubation at 30 °C, ATP (5 mm final con-
centration) and GroES (0.2 lm final concentration)
were added to the refolding mixture. The negative con-

trol for all these experiments comprise of the buffer
containing 0.08 m urea, which corresponds to the
residual concentration of urea in the refolding mixture,
and the positive control was the buffer containing
0.1 lm native MalZ protein and 0.08 m urea. 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 ª 2007 The Authors Journal compilation ª 2007 FEBS
chemical chaperone-mediated refolding was found to
be slower than the GroEL ⁄ GroES mediated refolding
process. By contrast, GroEL and GroES-assisted
refolding was faster, reaching the maximum refolding
yield (55%) within 1 h, with no further enhancement
in refolding recovery being observed after that

(Table 3). For example, the maximum recovery of
MalZ activity in the chemical chaperone-mediated pro-
cess (proline) was only 18% after 15 min, whereas
GroELS-assisted refolding produced a recovery of
34% after the same refolding period (Table 3).
Combined application of chemical and molecular
chaperones provides higher refolding of MalZ
The combined effect of chemical and molecular chap-
erones on the refolding yield of MalZ over their indi-
vidual contribution was also investigated. It was
observed that their joint application produced a rela-
tively higher yield of refolded MalZ for all cases,
except the combination of EG and GroELS.
It has already been demonstrated that chemical as
well as molecular chaperones assist individually in the
refolding of MalZ in vitro by preventing its aggrega-
tion, and produce a substantial amount of the physio-
logically active form of the protein. Urea-denatured
MalZ was allowed to refold in the presence of chemi-
cal chaperones, GroEL, GroES and ATP and the
refolding was monitored by withdrawing the refolding
mixtures at different time intervals followed by a MalZ
activity assay. The recovered MalZ activity was com-
pared with the control where 0.08 m urea was present
in native MalZ sample. The percentage of refolding of
MalZ was calculated and the result was compared with
the extent of refolding achieved from the processes
mediated by both types of chaperones (Fig. 5). In each
case, it was observed that the recovery of refolded
MalZ was substantial and that the GroEL⁄ GroES-

assisted refolding and recovery at the earlier stage of
dilution was relatively higher (Table 4). By contrast,
chemical chaperone based refolding was initially slow
and increased gradually (Table 3). When the yield of
osmolyte-assisted refolding of MalZ was analyzed after
5 h of refolding, it was observed that the highest incre-
ment of recovery was achieved in the presence of
dimethylsulfoxide when used together with GroEL and
GroES (Fig. 6). Glycerol, TMAO and proline also
enhanced the recovery of MalZ to a good extent when
used in the presence of GroEL and GroES. However,
EG, as an exception, could not improve the recovery
of folded MalZ even in the presence of GroEL and
GroES.
Table 3. The dependence of the refolding yield of MalZ from the urea-denatured state on refolding time in the presence of an optimized
concentration of osmolytes at 30 °C.
Time
(min) Spontaneous
Chaperone(s) used
Glycerol Dimethylsulfoxide TMAO Trehalose EG Proline GroELS
109 2 6 5 4 089 15
15 11 12 10 11 14 09 18 34
30 11 23 25 20 26 09 37 44
60 11 37 34 34 33 10 45 55
120 11 40 39 42 37 12 50 55
300 11 52 42 44 41 12 55 55
600 11 61 45 49 42 12 58 55
1200 11 61 45 49 42 12 58 55
0
10

20
30
40
50
60
70
80
% Refolding Yield of MalZ
ELS
-+ -+ -+ -+ -+ -+
MalZ
++ ++ ++ ++ ++ ++ ++
-+
Glycerol
++
dimethylsulfoxide
++
TMAO
++
Trehalose
++
Proline
++
EG
++

Fig. 5. Histogram showing the reconstitution of urea denatured
MalZ (0.1 l
M) after 5 h of refolding in the presence of chemical or
molecular chaperone as well as in the presence of both chaper-

ones. The extent of refolding was expressed as the percent of
activity recovered compared to the same amount of native MalZ.
Refolding was initiated by 100-fold dilution of denatured MalZ into
refolding buffer (20 m
M sodium phosphate, pH 7.0, 1 mM MgCl
2
,
5m
M dithiothreitol) containing the respective chemical or molecular
chaperone system, as well as both chaperones.
S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase
FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6005
Chemical chaperones protect MalZ from
denaturation and irreversible aggregation
during thermal stress
MalZ loses its native conformation and undergoes
aggregation during incubation at 42 °C. We investi-
gated whether osmolytes such as glycerol and dimeth-
ylsulfoxide could protect MalZ against thermal
aggregation in vitro. The native MalZ was incubated
at 42 °C in the absence or presence of optimized
concentrations of chemical chaperones and its activity
was measured at different time intervals. It was
observed that the chemical chaperones reduced the
aggregation of MalZ efficiently (Fig. 7). The highest
degree of thermoprotection of MalZ was achieved
throughout the whole incubation period in the
presence of 1 m of proline, which can be best com-
pared with glycerol-assisted protection because glycerol
also protected the denaturation of MalZ significantly

(Fig. 7). All of the chemical chaperones that were used
in the studies, with the exception of EG, protected
MalZ from thermal stress to a different extent.
Discussion
Six different chemical chaperones (glycerol, dimethyl-
sulfoxide, TMAO, EG, trehalose and proline) were
studied to investigate whether they assist in the
refolding of a 69 kDa E. coli monomeric protein
maltodextrin glucosidase. It was previoudly reported
that GroEL, in the presence of its cochaperonin
GroES, assists in the folding of MalZ in vivo and,
in the same study, it was also demonstrated that
GroEL, GroES and ATP together led to the sub-
stantial recovery of folded MalZ upon refolding
from a GdnHCl denatured state in vitro [33]. In the
Table 4. The effect on refolding of MalZ by the combined application of chemical chaperones and cellular chaperones when protein was
unfolded by 8
M urea at 30 °C.
Time
(min) Spontaneous
Chaperone(s) used
GroEL
+ glycerol
GroEL
+ dimethylsulfoxide
GroEL
+ TMAO
GroELS
+ trehalose
GroEL

+EG
GroEL
+ proline GroELS
1 09 10 11 9 10 11 12 15
15 11 30 28 31 19 13 26 34
30 11 39 34 37 28 14 34 44
60 11 47 44 45 36 14 46 55
120 11 54 52 50 43 14 56 55
300 11 65 60 55 47 14 61 55
600 11 71 62 61 48 14 68 55
1200 11 71 62 61 48 14 68 55
0
5
10
15
20
25
30
35
40
16.7%
17.2%
14.3%
24.5%
38%
28%
ProlineEGtrehaloseTMAODMSOGlycerol
Increase of MalZ refolding yield
Fig. 6. An optimized concentration of different chemical chaper-
ones was used along with GroEL ⁄ GroES in the assisted refolding

of MalZ at 30 °C. The increment of refolding yield of MalZ, due to
combined application of chemical chaperones and GroEL ⁄ GroES
over the yield where chemical chaperones were solely used as
folding aids during refolding, was calculated and expressed as a
percentage. DMSO, dimethylsulfoxide.
0
20
40
60
80
100
0 5 10 15 20 25 30
Time (min)
% Relative MalZ activity
Fig. 7. Thermoprotection of MalZ by glycerol, dimethylsulfoxide and
TMAO. Native MalZ was incubated in buffer (j), 3
M glycerol (d),
3
M dimethylsulfoxide (m), 3 M TMAO (.), proline (·), EG (+) and
Trehalose (r)at42°C. At different time intervals, aliquots were
withdrawn and enzymatic assay of MalZ was carried out.
Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al.
6006 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS
present study, chaperone-like activity was observed
for all of the above-mentioned osmolytes, except
EG, in the refolding of urea denatured MalZ.
GroEL, GroES and ATP-mediated refolding of urea
denatured MalZ was also performed and it was
observed that the refolding yield of MalZ was
enhanced by five-fold over the spontaneous yield,

which was only 11% at 30 °C (Table 3).
The above-mentioned observations indicate that
MalZ was unable to fold itself properly in vitro. Proba-
bly, the nascent polypeptide forms aggregate during its
refolding process and hence it requires the assistance of
GroEL and GroES in its productive folding. We were
particularly interested to use a few osmolytes, com-
monly known as ‘chemical chaperones’, in the refold-
ing studies of MalZ from its urea denatured state, to
examine whether those osmolytes could enhance the
in vitro folding of MalZ. Our findings demonstrated
that the above-mentioned osmolytes acted as chaper-
ones in the refolding of MalZ and enhanced the
in vitro refolding yield of MalZ significantly, with the
exception of EG, which did not improve the refolding
yield of MalZ over the spontaneous one.
Among all the osmolytes used, glycerol, which is
commonly known to be a strong stabilizer of the
native structure of proteins, led to a maximum
recovery of active MalZ during the refolding process
at a concentration of 2 m (Table 1). However, the
refolding yield of MalZ was reduced beyond 2 m con-
centration, suggesting that, in the osmolyte-mediated
refolding of MalZ, factors other than thermodynam-
ics also contribute significantly. A similar result was
observed in the proline-assisted refolding of MalZ.
Proline, at a lower concentration, has also been
reported to be an excellent protein folding chaperone
because it plays a chaperone-like role in the refold-
ing of many proteins [26,36–38]. Experimental evi-

dences have suggested that proline inhibits protein
aggregation not only by binding to folding inter-
mediate(s) and trapping the folding intermediate(s)
into enzymatically inactive, ‘aggregation-insensitive’
state(s), but also by accelerating the hydrophobic
collapse of creatine kinase to a packed protein
[38,39]. It has also been reported that, at higher con-
centrations (> 1.5 m), proline forms loose, higher-
order molecular aggregate(s). The supramolecular
assembly of proline is found to possess an amphi-
pathic character. Formation of higher-order aggre-
gates is believed to be crucial for proline to function
as a protein folding aid [39]. In the present study,
we also observed that, unlike glycerol, which even at
concentrations of 3–4 m led to a good recovery of
refolded protein, low concentrations of proline pro-
duced the highest refolding recovery, whereas, at a
higher concentration (> 1 m), the refolding yield
reduced drastically (Table 1). The reduction in the
efficiency of refolding might have been due to the
formation of a higher-order molecular aggregate ⁄
supramolecular assembly that stabilizes protein
aggregates. Other osmolytes (e.g. dimethylsulfoxide,
TMAO and trehalose) also provided a good yield of
refolded MalZ (Table 1) under similar conditions.
Unlike glycerol and dimethylsulfoxide, TMAO had
an optimum concentration of 3 m where the yield of
refolded MalZ was maximum. Unfortunately, EG
could not improve the reconstitution yield of MalZ
over spontaneous refolding.

Osmolyte-induced refolding efficiency was also
monitored at different temperatures in the range
10)40 °C (Table 2). With the exception of EG, all
other osmolytes and GroEL ⁄ GroES demonstrated
their ability to protect MalZ from thermal denatur-
ation and rescued a substantial amount of active
MalZ compared to spontaneous recovery. GroEL
and GroES are heat shock proteins known to pre-
vent other cellular proteins from thermal denatur-
ation. Glycerol and proline have also been reported
in many cases to prevent protein aggregation against
thermal stress and to stabilize the native structure of
proteins in solution. Indeed, GroEL ⁄ GroES showed
the highest protection by rescuing 66% of active
MalZ molecules from denaturation, whereas glycerol
and proline, among all osmolytes, provided compara-
ble protection (> 60%) against thermal denatur-
ation. Dimethylsulfoxide and TMAO produced
approximately 50% recovery, and trehalose offered
the lowest protection of approximately 40% at
40 °C. EG did not show any protection.
We were also interested to investigate the effect of
the combined application of osmolytes and GroELS
on the refolding efficiency of MalZ. Interestingly, for
all cases except EG, the yield was enhanced during the
chemical and molecular chaperone-assisted refolding
compared to the yield obtained from individual chap-
erone-assisted refolding (Fig. 5). This was most likely
due to osmolytes assisting in the local folding of
GroEL-bound non-native protein molecules. It was

also determined that the extent of refolding due to
GroEL ⁄ GroES alone was approximately 60% at
30 °C. This signifies that the remaining approximately
40% of MalZ molecules must have formed aggregates.
The other possibility is that those MalZ molecules not
captured by GroEL, or that received no productive
assistance from GroEL, would be taken care of by
osmolytes and that is why there was a significant
increase of refolding yield when both osmolytes and
S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase
FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6007
GroELS were used together. As shown in Fig. 5, we
also observed that the percent increase of refolding
yield of MalZ in dimethylsulfoxide and GroELS-
assisted folding was maximum among all cases, except
for trehalose where it was lowest. Perhaps the viscos-
ity factor is important here as trehalose has the highest
viscosity, and this might have prevented GroEL from
binding to unfolded MalZ molecules in solution.
To observe the effect of chemical chaperone-medi-
ated thermoprotection on the activity of MalZ, the
MalZ solution temperature was increased to 42 °C. We
observed that there was gradual loss of activity with
time in the absence of any osmolytes in solution and,
after 15 min of incubation time, complete biological
activity of the protein was lost. However, when various
osmolytes were added to the MalZ solution and incu-
bated at the higher temperature, the enzymatic activity
was protected significantly, even after 30 min (Fig. 7).
In conclusion, polyol osmolytes, glycerol, dimethyl-

sulfoxide, TMAO, trehalose, EG and proline assist in
the productive refolding of MalZ considerably. There
was a good correlation between the increase in the
refolding yield in the presence of osmolytes and its
ability to suppress protein aggregation. The decrease in
the refolding yield at high osmolyte concentrations
suggests that, in addition to the thermodynamic con-
siderations, kinetic factors also are important during
refolding in the cosolvents. There should be a cut-off
viscosity of the refolding medium in the presence of
the respective osmolyte that may lead to productive
folding of MalZ. GroEL ⁄ GroES and osmolytes in
combination enhanced the refolding yield of MalZ to
the higher level. The findings also suggest that small
organic molecules such as osmolytes can be used as
effective agents in preventing protein aggregation and
in the therapy of several aggregation-related debilitat-
ing diseases when applied together with molecular
chaperones such as GroEL and GroES.
Experimental procedures
Materials
TG1, M15 and BL21 E. coli strains were used for the expres-
sion and purification of MalZ, GroEL and GroES, respec-
tively. The plasmid pCS19MalZ containing (His)6malZ
was generous gift from W. Boos (University of Konstanz,
Germany) and the plasmid pACYCEL, containing the
groEL gene, and pET22dES, containing the groES gene,
were a gift from A. L. Horwich (Yale University, CT,
USA). Urea, glycerol, TMAO, dimethylsulfoxide, trehalose,
ethylene glycol, proline and p-nitrophenyl-d-maltoside were

purchased from Sigma Chemical Company (St Louis, MO,
USA) and the disodium salt of ATP was purchased from
Sisco Research Laboratories Pvt. Ltd (New Delhi, India).
Dithiothreitol and MgCl
2
were purchased from Merck (New
Delhi, India). All other reagents used were of analytical
grade.
Purification of MalZ, GroEL and GroES
MalZ and GroEL were overexpressed in TG1 and M15
E. coli cells, respectively, and GroES was overexpressed in
BL21 E. coli cells. Plasmids pCS19MalZ, pACYCEL and
pET22dES were used for overexpression of MalZ, GroEL
and GroES, respectively. Chaperones were purified as previ-
ously described [39,40]. Cells were disintegrated in French
press and lysates were centrifuged at 23 500 g for 45 min.
Supernatant was separated and applied for chromato-
graphic process in an AKTA FPLC system (Pharmacia,
USA). GroEL was purified using FFQ anion-exchange
chromatography (Pharmacia, USA), GroES was purified
using FFSP cation-exchange chromatography (Pharmacia).
After the FPLC purification process, affigel blue treatment
was performed on GroEL to remove bound substrate pro-
teins and, for GroES, differential precipitation by lowering
the pH of the protein mixture was applied to eliminate
other proteins before the FPLC purification process. MalZ
purification was a single step process using Ni-chelating col-
umn HisTrap HP (Pharmacia).
Denaturation of MalZ
MalZ was unfolded in the 20 mm sodium phosphate buffer,

pH 7.0, containing 8 m urea and 20 m m dithiothreitol. The
denaturation was confirmed by observing the complete loss
of enzyme activity of MalZ in solution and also from the
maximum change in the intensity of intrinsic tryptophan
fluorescence emission.
Tryptophan fluorescence studies
Steady state fluorescence was recorded on a LS55 lumines-
cence spectrofluorimeter (Perkin-Elmer, New Delhi, India).
Intrinsic tryptophan fluorescence spectra were recorded by
exciting the samples at 279 nm with excitation and emission
slit widths set at 5.0 and 7.5 nm, respectively. The emission
spectra were recorded in the range 300–400 nm. Baseline
corrections were carried out using buffer without protein in
all the cases.
Refolding of MalZ
MalZ (10 lm) was denatured in 20 mm sodium phosphate
buffer, pH 7.0, containing 8.0 m urea and 20 mm dithio-
erythritol and incubated for 4 h at 30 °C. Refolding experi-
ments were carried out by diluting the denatured protein
Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al.
6008 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS
100-fold into the refolding buffer (20 mm sodium phos-
phate, pH 7.0, 1 mm MgCl
2
and 5 mm dithiothreitol). The
enzyme concentration during refolding was 0.1 lm. In case
of cosolvent-assisted refolding, various cosolvents, such as
glycerol, dimethylsulfoxide and TMAO, were added at the
desired concentrations to the refolding buffer into which
the denatured protein was diluted. Refolding mixtures were

incubated at the desired temperatures until completion of
the reaction. The extent of refolding was calculated from
the recovered activity relative to the activity of the same
amount of native protein.
Acknowledgements
The authors thank DST, Government of India, and
MHRD, Government of India, for financial support.
We also thank Professor Winfried Boos, University of
Konstanz, Germany, for the gift of plasmids encoding
His-tagged MalZ and Professor Arthur Horwich, Yale
University, CT, USA, for providing the plasmids for
expressing GroEL and GroES.
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