The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

Co-expression of chaperonin GroEL/GroES enhances in vivo folding

of yeast mitochondrial aconitase and alters the growth
characteristics of Escherichia coli
Parul Gupta, Nishtha Aggarwal, Pragya Batra,
Saroj Mishra, Tapan K. Chaudhuri
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi,
Hauz Khas, New Delhi 110016, India
Received 24 January 2006; received in revised form 19 May 2006; accepted 23 May 2006
Available online 2 June 2006

Abstract
Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL
and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However,
preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins in
E. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence of
recombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependent
growth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. coli
chaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of coexpressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroEL & GroES together compensate this
reduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 C when compared with the M15 E. coli
cells, however, there is an increase of 20% at 37 C indicating the participation of endogenous chaperonin in the folding of a fraction
of over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cells
was enhanced by 30% at 37 C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase.
Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.
2006 Elsevier Ltd. All rights reserved.
Keywords: Over expression of recombinant aconitase; In vivo protein folding; Escherichia coli growth profile; Bacterial chaperonin GroEL and
GroES

1. Introduction
Abbreviations: AMPR , ampicillin resistance; LB, luria broth;
APS, ammonium persulfate; DTT, 1,4-dithiothreitol; HEPES, 4-2hydroxyethyl-1-piperazineethanesulfonic acid; IPTG, isopropyl -dthiogalactoside; SDS-PAGE, sodium dodocyl sulfate-polyacrylamide
gel electrophoresis
Corresponding author. Tel.: +91 11 2659 1012;
fax: +91 11 2658 2282.
E-mail address: tapan@dbeb.iitd.ac.in (T.K. Chaudhuri).
1357-2725/$ see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2006.05.013

The production of recombinant protein in Escherichia

coli is one of the major efforts in biotechnology today. A
major limitation in the over expression of recombinant
proteins is the inability of many recombinant polypeptides to fold into their biologically active conformations
within the milieu of the bacterial cell. The question of
protein folding has been a subject of intensive research

1976

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

since Anfinsen (1973) showed that a denatured protein could fold unassisted under in vitro conditions. In
vivo protein folding is a different phenomenon, complicated by macromolecular crowding in cytosol. The
mechanism of in vivo protein folding remains one of the
most intriguing problems to be elucidated in molecular biology (Cortazzo et al., 2002). The in vivo folding
pathways are affected by a number of factors, such as
physico-chemical conditions of the cellular environment
and transient interactions with other co translated proteins, not present in the simplified in vitro assays. In the
complex medium of the cell, the physical conditions of
temperature, pH, etc., are restricted, and the concentration of macromolecules is high creating a dynamically
changing environment for newly synthesized proteins
(Johnson & Craig, 1997). Molecular chaperones are a
class of proteins thought to facilitate protein folding in
this environment (Horwich et al., 1993; Johnson & Craig,
1997; McLennan & Masters, 1998). The discovery of
these helper proteins in the last decade has extended the
field of in vivo protein folding enormously. As unfolded
polypeptide contains many more exposed hydrophobic
residues than the polypeptide in its native state, they
are much more susceptible to aggregation. Whether the
polypeptide is a nascent chain on a ribosome or a mature
protein recently unfolded due to stress, suppression of
aggregation is essential in order to maintain proteins
in a state competent for folding. Molecular chaperones
are large family of proteins found in all types of organisms and have a very important role in protein folding
and maintaining protein homeostasis (Wong & Houry,
2004). Chaperone families are highly conserved across
genomes, suggesting that their functions are essential for
cellular life (Jordan et al., 2002). Chaperones are thought
to prevent newly synthesized proteins from misfolding and aggregating, impeding undesired hydrophobic
interactions, and allowing alternative folding pathways
(Hartl, 1996). They bind to the exposed hydrophobic
regions of nonnative proteins, hindering aggregation
(Hartl & Hayer-Hartl, 2002). Therefore, through regulated cycles of peptide binding and release, chaperones
facilitate the acquisition of the active conformation of
the polypeptides. The most extensively studied chaperones are the chaperonin GroEL and GroES from
E. coli (Fenton & Horwich, 1997). The atomic structure of GroEL (Braig et al., 1994) and GroES (Hunt et
al., 1996) are known and also that of the GroELGroES
complex formed in the presence of ADP (Xu et al., 1997).
Over expression of the molecular chaperones in E. coli
(Amrein et al., 1995; Caspers et al., 1994; Goloubinoff
et al., 1989; Lee & Olins, 1992; Nishihara et al., 2000)
for obtaining large quantities of correctly folded protein

of interest is a common method, making their downstream processing much easier. The main purpose of
recombinant protein expression is often to obtain an
accumulation of high degree of soluble product in the
bacterial cell. This strategy is not always accepted by
the metabolic system of the host and in some situations
a cellular stress response is encountered (Srensen &
Mortensen, 2005).
In the last decade, extensive amount of work has
been done on the co-over expression of the GroEL/ES
in E. coli along with other foreign proteins. The co-over
expression of the bacterial chaperone system GroEL/ES
along with several proteins like -crystallin (Goenka
& Mohan Rao, 2001), malate dehydrogenase (Ranson
et al., 1995), medium-chain acyl-CoA dehydrogenase
(MCAD) (Bross et al., 1993), carbamoylase (Sareen et
al., 2001) and aconitase (Chaudhuri et al., 2001) significantly enhance the yield of soluble protein. The
effect of GroEL/ES on protein folding in association
with other chaperones like trigger factor (Nishihara
et al., 2000; Vorderwlbeckea et al., 2004) and DnaK
(Vorderwlbeckea et al., 2004) has also been studied. It
has been found that the over expression of GroEL/ES
restores appropriate protein folding in the cells where
trigger factor and DnaK have been deactivated. Extensive work on the mechanistic aspects of protein folding
in presence of chaperonin has also revealed valuable
information on protein folding pathway and aggregation
(Chaudhuri et al., 2001; Farr et al., 2003; Jewett et al.,
2004; Lin & Rye, 2004; Ueno et al., 2004). However,
studies on the effect of co-expressing chaperonin in the
cell along with other proteins and their implications on
the cell growth have not been thoroughly investigated.
Parameters like optimum temperature, inducer concentration, duration of induction, etc., play an important
role in enhancing the level of production of the desired
protein in its native form. Here we have studied different aspects of cell growth parameters during the over
production of recombinant aconitase in presence and
absence of over producing exogenous molecular chaperonin, GroEL and GroES. Our main aim is to understand
the conditions for optimum production of recombinant
aconitase in the presence and absence of co-expressed
chaperonin GroEL and GroES in E. coli and subsequent
growth profiles and their temperature dependence.
2. Materials and methods
2.1. Chemicals and reagents
Luria broth (LB) for E. coli growth and antibiotics
kanamycin, ampicillin and tetracyclin were obtained

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

1977

from HiMedia (India). 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 1,4-dithiothreitol

(DTT), acrylamide, bis-acrylamide, standard molecular
weight markers, ammonium persulfate (APS) and
isopropyl -d-thiogalactoside (IPTG) were obtained
from Bangalore, Genei (India). Other reagents and
chemicals used were from Merck (Germany) and Sigma
(USA).

2002). Expression was checked by running various samples on 15% SDS-PAGE (Laemmli, 1970; Sambrook &
Russell, 2001). To study the effect of temperature on
the specific growth rate, the experiment was carried out
at two different temperatures (25 C and 37 C). Effect
of induction on the growth rate of various strains was
observed from the growth curve generated with and without induction.

2.2. Strains and plasmids

2.4. Determination of the specic growth rate

constants for bacterial growth

The gene for yeast mitochondrial aconitase, cloned

in the pQE60 vector from Qiagen (AMPR selectable
marker) with ColE1 origin of replication was obtained
from Dr. Sabine Rospert, Germany. The constructs
pACYCEL over expressing GroEL and pACYCELS
over expressing GroEL and GroES (with tetracycline
resistance) are generous gift from Dr. Arthur L. Horwich,
USA. M15 E. coli strain, K12 derivative was used for the
expression of various plasmids. M15 strain, containing
multiple copies of pREP4 plasmid, was maintained in
presence of kanamycin. pREP4 plasmid carries the lacI
gene encoding the lac repressor and hence the expression of aconitase in pQE60 is regulated. M15 cells were
transformed with plasmid pAco to express only aconitase, with pAco and pACYCEL to express aconitase and
GroEL, with pACYCELS to express GroE-GroES and
with pAco and pACYCELS to express aconitase, GroEL
and GroES. The antibiotic concentration used for the
optimum growth of the cells was 25 g/ml, 80 g/ml and
12.5 g/ml for kanamycin, ampicillin and tetracycline,
respectively.
E. coli M15 strain was activated from a stab culture by streaking on luria agar plate supplemented with
required amount of antibiotics. Ten milliliters LB supplemented with antibiotics was inoculated and cultured
at 37 C. This strain was further maintained by making 20% glycerol stocks, frozen in liquid nitrogen and
stored at 80 C. These stocks were used subsequently
for making competent cells and transformation with various plasmids. Various transformed recombinant E. coli
cells were also grown and maintained as described above.
2.3. Determination of growth prole
Various E. coli strains were grown at 25 C and
at 200 rpm in shake flasks and 1 ml of cell suspension was withdrawn at various time intervals for
turbidity measurements at 650 nm using UVIKON 930
spectrophotometer (Kontron instruments, USA). Induction was done at OD650 of 0.71.0 with 100 M IPTG
for expression of aconitase (Christodoulou & Vorgias,
37 C,

The specific growth rate constants for various growth

profiles was calculated by plotting absorbance versus
time and obtaining the slope by exponential trend using
the following equation:
X = X0 expt
where X = biomass at time t; X0 = biomass at time
t = 0; = specific growth rate constant; t = time in
hours.
2.5. Estimation of the relative intensities of the
bands in the SDS gel
BioRad (USA) gel documentation unit was used for
estimating the relative quantities of the various protein
bands observed on the gel. Gel image was taken and the
various lanes were framed using manual frame lanes
toolbar. The number of lanes in the frame was kept the
same as that present in the gel. The lanes drawn were
adjusted to fit the size of the lanes in the gel. Lane
background subtraction was carried out to remove the
background intensity of the gel itself from the bands.
Band analysis quick guide from the Quantity one program (BioRad) was used to select the bands. Bands were
detected using band detect option and the area of the
band with the required peak was adjusted using adjust
band option to get the region of the band to be estimated.
Relative quantity of the band selected was measured by
selecting relative quantity from band attributes toolbar. Relative quantity of a particular band is the quantity
measured by its intensity, expressed as a percentage of
the total intensity of all the bands in the lane.
2.6. IPTG titration
Various E. coli strains transformed with recombinant
plasmids were grown in LB medium supplemented with
antibiotics upto OD650 between 0.6 and 1.0. At this point
each culture was divided into nine parts of 10 ml each.
IPTG was added at nine different concentrations ranging

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P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

from 0 M to 200 M (Christodoulou & Vorgias, 2002).

The cultures were left overnight at 37 C and the level
of expression were analyzed by 15% SDS-PAGE.
2.7. In vivo folding of aconitase
Amount of folded protein in a cell can be estimated
based on the principle that the proteins with a threedimensional structure are soluble in the cytoplasm and
in aqueous buffer, however, denatured proteins are insoluble and occur as aggregates (Chaudhuri et al., 2001).
Thus, to estimate the extent of correct folding of aconitase in vivo, the induced cells were pelleted and broken
down by sonication to release the intracellular components in the lysis buffer. Normalization of the cell culture
was done, such that same number of cells were taken for
the analysis of each sample. The soluble components
were separated from the insoluble mass by centrifugation of the cell lysate. The supernatant and the pellet
were analyzed separately for the presence of aconitase by
SDS-PAGE and enzymatic activity test. Culture broths of
different transformed strains expressing aconitase at different temperatures were harvested and resuspended in
lysis buffer containing 50 mM HEPES (pH 7.4), 0.5 mM
MgCl2 , 1 mM DTT (Chaudhuri et al., 2001). These cells
were disrupted using ultrasonicator, followed by centrifugation at 10,000 rpm for 45 min. The supernatant and
pellet were separated and freeze dried in a lyophillizer
(LABCONCO freezedry 4.5). Lyophilized supernatant
and pellet were resuspended in the loading buffer and
analyzed by SDS-PAGE. Aconitase activity assay was
done taking 50 g of total protein in each case.
2.8. Aconitase assay
Aconitase activity was quantitated using a coupled
enzyme assay. Aconitase catalyses conversion of citrate to isocitrate, which in turn is converted to -keto
glutarate in presence of isocitrate dehydrogenase along
with the formation of NADPH from NADP (Morrison,

1954). The assay was performed by taking 2050 g of

protein in a 1 ml reaction volume (0.1 M TrisHCl pH
8, 0.66 mM sodium citrate, 0.66 mM MnSO4, 0.5 mg/ml
-NADP and 0.17 mg/ml isocitrate dehydrogenase). The
formation of NADPH was monitored at 340 nm using
time/kinetics application on Beckman Coulter DU 800
(USA).
3. Results
3.1. Changes in growth prole of E. coli cells on
transformation with various recombinant plasmids
E. coli is used extensively for the expression and over
production of both prokaryotic and eukaryotic recombinant proteins, as it requires very simple growth conditions. The growth profile of E. coli changes considerably depending on the type and number of recombinant
plasmid it contains and the number of proteins it over
expresses. Bacterial cell growth was monitored by measuring turbidity of the cell culture at 650 nm using a
spectrophotometer.
3.1.1. Effect of plasmid characteristics on the
growth rate of transformed cells
The growth curve for various transformed strains of E.
coli namely, M15 cells expressing aconitase, M15 cells
expressing aconitase and GroEL, M15 cells expressing
aconitase, GroEL and GroES and M15 cells expressing
GroEL and GroES were generated by plotting turbidity values at 650 nm of various strains against growth
time without induction. M15 strain was used as a negative control in all these studies. The growth profiles of
transformed cells without induction showed the effect
of plasmid replication and maintenance on the rate of
growth (Table 1). At both the temperatures (25 C and
37 C), cells containing pACYCEL plasmid showed a
decrease of more than 25% in the growth rate. At 25 C,
presence of pACYCELS plasmid in M15 cells reduced
the growth rate by about 40% as compared to M15 wild

Table 1
Specific growth rate constants () for the growth of M15 E. coli strain under various plasmid-containing situations at 25 C and 37 C
E. coli strain

M15 wild type

M15 + Aco
M15 + Aco + GroEL
M15 + GroEL + GroES
M15 + Aco + GroEL + GroES

at 25 C

at 37 C

Uninduced strains

Induced strains

Uninduced strains

Induced strains

0.12(0.985)
0.11(0.9827)
0.08(0.9806)
0.07(0.9874)
0.07(0.9957)

0.10(0.9908)
0.07(0.9947)
0.08(0.9877)
0.07(0.9869)
0.07(0.9971)

0.20(0.996)
0.20(0.9942)
0.15(1.000)
0.22(0.9949)
0.21(0.9999)

0.17(0.9981)
0.20(0.9938)
0.20(0.9985)
0.18(0.9926)
0.22(0.9932)

Regression coefficient values are given in brackets.

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

type cells, whereas, at 37 C, presence of pACYCELS

plasmid showed an increase in growth rate by about 10%
(Table 1). No appreciable change in the growth profile was observed for M15 cells on transformation with
aconitase plasmid (Table 1).
3.1.2. Effect of IPTG induction on the growth
prole of various transformed strains at different
temperatures
Growth profiles of all the M15 strains transformed
with pAco, pAco-pGroEL, pGroELS, and pAcopGroELS were studied with and without IPTG induction.
Induction was carried out with 100 M IPTG in the turbidity range (OD650 ) of 0.6 to 1.0. At 25 C, all aconitase
over expressing M15 strains, with and without recombinant GroEL/ES showed a reduction in the growth rate,
the most notable however, are the M15 cells harboring
only aconitase, which showed a 30% reduction in growth
rate (Table 1). At 37 C, a complete reversal in the growth
profile was observed. Aconitase over expressing M15
strains, showed an overall rise in growth rate after induction. Only aconitase over expressing strain showed a rise
of 20% when compared with wild type M15 cells and
20% increase in growth rate was observed for aconitase
and GroEL over expressing strain (Table 1). A significant rise of 30% was observed in the specific growth rate
of M15 cells expressing aconitase in presence of both
GroEL and GroES. The changes in specific growth rate
of various cultures on induction are unavoidable, however, such changes can be minimized by redesigning and
engineering various pathways (Flores et al., 2004).
3.1.3. Effect of temperature on the growth prole of
various transformed strains after induction with
IPTG
Growth profiles of different transformed M15 cells
containing pAco, pAco + pGroEL, pAco + pGroELS and
pGroELS plasmids were studied at two different temperatures, 37 C and 25 C, after induction with IPTG.
At 37 C, the growth rate for all transformed cells was
found to be about two times higher than those at 25 C for
the same strain. Other changes in the trends for various
growth parameters for different transformed cells have
been observed by changing temperature (as discussed in
the above result Sections 3.1.1 and 3.1.2).
3.1.4. Over expression of aconitase in M15 cells
with time of induction in the absence and presence
of over expressing chaperonin GroEL/ES
The gene for yeast mitochondrial aconitase (pAco) is
cloned in pQE60 vector, which confers ampicillin resistance to the cells and has ColE1 origin of replication. The

1979

presence of pREP4 plasmid in the M15 E. coli strain provides the lacI gene from the Lac operon, which represses
the aconitase expression in pQE60. Addition of inducer
to the cell culture medium activates the expression of the
aconitase. A kinetics analysis, after addition of IPTG,
was done to estimate the duration of induction required
for the expression of aconitase in presence and absence of
chaperonin at 25 C and 37 C. The expression of aconitase was estimated by SDS-PAGE analysis of samples
withdrawn at different time intervals (Figs. 1B and 2B). It
was found that at 37 C, aconitase expression in absence
of chaperonin requires 5 h of incubation after induction
(Fig. 1A). In presence of only GroEL it requires the least
time of 2 h for induction, whereas, when both GroEL and

Fig. 1. (A) Relative expression of aconitase with time in the presence

and absence of GroEL/ES at 37 C. The graph shows the different
relative intensities of the aconitase bands in the gel depicting the levels of expression of aconitase in presence and absence of GroEL and
GroEL/ES at 37 C with different durations of incubations. The first
bar of the triad shows only aconitase expression, the second bar of
the triad shows aconitase expression in presence of only GroEL and
the third bar of the triad shows aconitase expression in presence of
both GroEL and GroES. (B) 15% SDS-PAGE shows level of aconitase
expression in presence and absence of GroEL and GroEL/ES at 37 C
with different durations of incubations. Lane 2, 0 h; lane 3, 2 h; lane
4, 5 h; lane 5, 7 h; lane 6, 9 h; lane 7, 11 h and lane 8, 15 h. Lane 1
shows medium range standard molecular markers. Top panel shows
over expression of aconitase only, center pane shows aconitase over
expression in presence of GroEL and bottom panel shows aconitase
over expression of aconitase in presence of GroEL and GroES.

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P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

Fig. 2. (A) Relative expression of aconitase with time in the presence

and absence of GroEL/ES at 25 C. The graph shows the different
relative intensities of the aconitase bands in the gel depicting the levels of expression of aconitase in presence and absence of GroEL and
GroEL/ES at 25 C with different durations of incubations. The first
bar of the triad shows only aconitase expression, the second bar of
the triad shows aconitase expression in presence of only GroEL and
the third bar of the triad shows aconitase expression in presence of
both GroEL and GroES. (B) 15% SDS-PAGE shows level of aconitase
expression in presence and absence of GroEL and GroEL/ES at 25 C
with different durations of incubations. Lane 2, 0 h; lane 3, 3 h; lane
4, 5 h; lane 5, 10 h; lane 6, 13 h; lane 7, 14 h and lane 8, 17 h. Lane
1 shows medium range standard molecular markers. Top panel shows
over expression of aconitase only, center pane shows aconitase over
expression in presence of GroEL and bottom panel shows aconitase
over expression of aconitase in presence of GroEL and GroES.

GroES are present, expression of recombinant aconitase

requires much longer induction period of 11 h. At 25 C,
expression of aconitase requires 13 h of incubation after
induction in absence of chaperonin (Fig. 2A ). M15 cells
expressing aconitase and GroEL requires 10 h and aconitase expression in presence of both chaperonin requires
14 h of incubation after induction.
3.1.5. Inducer concentration required for optimum
expression of recombinant aconitase
M15 cells transformed with pAco in presence and
absence of pGroEL/pGroELS were induced with different concentration of IPTG, when the turbidity of the
culture at 650 nm reached to 0.9 and were left at 37 C

Fig. 3. (A) Optimization of IPTG concentration for aconitase expression. Variation of expressed aconitase with change in inducer concentration. Solid squares show M15 cells expressing aconitase only,
solid triangles show M15 cells expressing aconitase and GroEL, solid
circles show M15 cells expressing aconitase, GroEL/GroES. Optimum IPTG required for induction of aconitase is 75 M (Origin 5.0
software was used to fit the graph). (B) 15% SDS-PAGE showing
variation of expressed aconitase with change in IPTG concentration.
Lane 1, standard protein molecular weight markers. Lane 2, 0 M
IPTG. Lane 3, 10 M IPTG. Lane 4, 20 M IPTG. Lane 5, 50 M
IPTG. Lane 6, 75 M IPTG. Lane 7, 100 M IPTG. Lane 8, 125 M
IPTG. Lane 9, 150 M IPTG. Lane 10, 200 M IPTG. Top panel
shows induced aconitase in absence of chaperonin, second panel shows
induced aconitase expression in presence of GroEL, third panel shows
induced aconitase expression in presence of GroEL and GroES and
bottom panel shows constitutive expression of GroEL and GroES in
cells expressing no aconitase.

for 24 h. No over expressed protein was found in M15

cells on IPTG induction. GroEL and GroES were found
to be over expressed constitutively and did not require
any inducer for complete expression (Fig. 3B). Recombinant aconitase expression was found to be inducible by
IPTG. M15 cells expressing pAco gene in the absence
and presence of chaperonin was found to require 75 M
IPTG for optimum expression of aconitase (Fig. 3A).
3.2. Over expression of aconitase in E. coli M15
cells in presence and absence pGroEL/pGroELS
E. coli M15 strain containing pREP4 plasmid was
transformed with high copy number plasmid (pAco) con-

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

1981

Table 2
Expression level of aconitase in presence and absence of GroEL/ES at
25 C and 37 C

Fig. 4. (A) Expression levels of aconitase in the presence of GroEL and

GroEL/ES. Graph shows changes in the level of expression of aconitase in recombinant M15 cells expressing aconitase in absence and
presence of GroEL and GroES (relative band intensities of aconitase
have been compared in various cases). Bold check shows the variation at 25 C and hollow check shows at 37 C. (B) 15% SDS-PAGE
showing changes in the level of expression of aconitase in recombinant
M15 cells, expressing aconitase in absence and presence of GroEL and
GroES. Standard molecular markers in lane 1 and lane 2 show uninduced aconitase, lane 3 and 4 show induced aconitase in duplicate,
lane 5 show uninduced aconitase in presence of GroEL, lane 6 and
lane 7 show induced aconitase in presence of GroEL and lane 8 shows
uninduced aconitase in presence of GroEL and GroES, lane 9 and lane
10 show induced aconitase in presence of GroEL and GroES.

taining the gene for the yeast mitochondrial aconitase

with a Lac-regulated promoter. A distinct band in SDSPAGE observed in the transformed strain clearly showed
the over expression of aconitase in the strain (Fig. 4B).
E. coli M15 strain containing pACYCEL plasmid constitutively over expressing GroEL was transformed with
pAco and the expression of aconitase showed a marked
reduction of about 40% at 37 C (Fig. 4A). Whereas,
when pAco is expressed in presence of pGroELS the
reduction in aconitase expression is only 20%. The
presence of the gene for a large 800-kDa protein in
pGroEL/pGroELS, directs most of the energy produced
by metabolism in the cell for over production of GroEL,
resulting in a reduced aconitase expression in the cell.
Presence of both GroEL and GroES in the cell helps to
fold the over expressed aconitase correctly. At 25 C,
almost no change in the expression in the aconitase is
observed in presence of GroEL/GroES (Table 2 and
Fig. 4A).

Strain

Relative quantity of
aconitase at 25 C

Relative quantity of
aconitase at 37 C

Aconitase
Aconitase with GroEL
Aconitase with GroEL
and GroES

3.5
3.4
3.3

5.9
3.9
4.9

3.2.1. In vivo folding of aconitase in absence and

presence of co-expressed GroEL and GroES in E.
coli cells
Non-native aconitase lodges itself as insoluble aggregates in a cell, which is deficient in chaperonins, as
compared to being fully soluble in a wild type cell producing both the chaperonin (Dubaquie et al., 1998). The
extent of correctly folded native protein in a cell can
be determined based on the principle that, a folded proteins would be soluble, whereas the denatured proteins
would form aggregates and stay insoluble. On cell disruption and fractionation, the supernatant contains the
soluble proteins and all the aggregated proteins along
with cell debris form a pellet. SDS-PAGE analysis of
the supernatant and pellet fraction of the cell lysate
(Fig. 5B) showed that, in the absence of exogenous
GroEL and GroES, 35% of the over expressed aconitase was found in the soluble form in the supernatant at
25 C (Fig. 5A) which increased to 40% at 37 C. In
presence of chaperonin GroEL, only 25% of the over
expressed aconitase showed up as folded fraction in
SDS-PAGE at 25 C, whereas 20% of the over expressed
aconitase appeared to be folded at 37 C. When both
the chaperonin GroEL and GroES were present approximately 50% of expressed aconitase was soluble at 25 C
and about 40% at 37 C (Fig. 5A). Aconitase activity
data (Fig. 5C) show that the soluble form of aconitase is
also biologically active. When pGroEL alone is present
in a cell, which is over expressing aconitase, aconitase
gets trapped in the hydrophobic cavity of GroEL; hence
it neither folds nor gets released as a folding competent
intermediate. GroEL remains in the fully native form in
the cell and hence appears in the supernatant. The proteins that remain bound with GroEL will appear in the
supernatant, even if they are not biologically active or
in a folded state. Thus, some GroEL bound non-native
aconitase appears in the soluble form with GroEL alone
when tested by SDS PAGE (Fig. 5B). The increase in the
amount of folded aconitase in presence of GroEL and
GroES clearly shows that presence of both GroEL and
GroES are required for the correct folding of aconitase.

1982

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

These observations are consistent with the earlier observations by Dubaquie et al. (1998) that aconitase does not
fold correctly on inactivation of the chaperonins in yeast
mitochondria.
4. Discussion

Fig. 5. (A) In vivo folding of aconitase in the absence and presence of

GroEL/ES. Graph shows the change in the level of folded aconitase in
the presence and absence of GroEL and GroEL/ES at 25 C and 37 C
in vivo. Bold waves show folding at 25 C and hollow waves shows
folding at 37 C. (B) 15% SDS-PAGE shows the change in the level
of folded aconitase in vivo, in the presence and absence of GroEL and
GroEL/ES at 25 C and 37 C. First column of gels depicts folding at
25 C and the second column shows folding at 37 C. Total amount of
aconitase (whole cell) in the first lane, folded aconitase (supernatant) is
seen in the central lane and aggregated aconitase (pellet) is in the third
lane. Amount of folded aconitase in presence of GroEL and GroES can
the seen the most distinctly in the central panel. Aconitase in absence
of any chaperonin is shown in the top panel and aconitase in presence
of only GroEL is in lowest panel (wc, whole cell; s, supernatant;

Cells derive energy for growth from metabolic activities. They use this energy to carry out essential functions like reproduction, growth, synthesis of various biomolecules like proteins and nucleic acid, wear and tear
management, etc. When a cell is programmed to produce
large amount of one or more recombinant proteins, the
inherent energetics of the cell growth gets disturbed. The
enhanced metabolic load exerted on the cell for maintenance and expression of recombinant plasmid may
adversely affect the rate of growth of a cell producing recombinant proteins as compared to the wild type
strain (Glick, 1995). Reduction in specific growth rates in
presence of a large number of plasmid in a recombinant
cell is well known (Milnitsky et al., 1997; Kobayashi et
al., 2002; Lee & Moon, 2003). However, in our study
we found varying trends. Transformation of M15 E.
coli strain with pACYCEL/ES increases the growth rate
significantly at 37 C, whereas it decreases at 25 C.
Higher temperature acts as an inducer for production of
heat shock proteins like GroEL and GroES. Enhanced
GroEL/ES production at 37 C helps the cells in preventing aggregation, which enhances the cell efficiency
and induces the correct folding of various proteins in the
cells resulting in enhanced growth. At 25 C, aggregation of both native and recombinant proteins is much less
in comparison to 37 C. Thus, at 25 C, the advantage
of over expressed GroEL and GroES becomes a liability
where it has to spend a lot of energy for synthesis of large
800 kDa GroEL (Fenton & Horwich, 1997), resulting in
reduction of the growth rate in presence of chaperonin.
Only pAco containing cells seemed to have had a very
little effect on the growth rates of uninduced cultures.
As M15 cells contain endogenous aconitase as well
as GroEL and GroES, hence, on transformation with
pAco and pGroELS, the cells will produce recombinant
aconitase and chaperonin along with their endogenous
counterpart. Enhanced production of heat shock proteins
at elevated temperatures in various organisms like E. coli
(Kusukawa & Yura, 1988; Rosen & Ron, 2002; Yura et

p, pellet). (C) Aconitase activity assay for over expressed aconitase

alone, with GroEL/ES and with GroEL alone are done and shown as
units per mg of protein per min. Black bars show activity at 25 C and
white bars at 37 C.

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

al., 1993), parasites (Toye & Remold, 1989) and brown

trout (Biswas & Sharma, 1994; Burkhardt-Holm et al.,
1998) are well documented. Endogenous chaperonin,
being heat shock proteins are activated at higher temperatures. Thus, on IPTG induction, all aconitase over
expressing strain shows an increase in the growth rate
at 37 C. The endogenous chaperones present in E. coli
produced at higher rates at 37 C may help a fraction of
the over expressed aconitase to fold correctly, which in
turn enhances the TCA cycle and generates more energy
resulting in enhanced growth rate. Infact, the enzymatic
activity test reveals higher aconitase activity in pAco
containing cells at 37 C (Fig. 5C). Co-expression of
GroEL along with aconitase at 37 C showed a similar
trend as that of only aconitase over expressing cells. A
major chunk of over expressed aconitase gets trapped
in GroEL cavity (Chaudhuri et al., 2001) and in the
absence of adequate GroES, it is unavailable for both
folding by active endogenous chaperonin and aggregation. Growth rate is maximum when the entire folding
machinery is present since: (a) larger amounts of both
GroEL and GroES help in correct folding of various
proteins, enhancing cell efficiency by minimizing toxic
effects of protein aggregation, and (b) the extra energy
required for the expression and synthesis of GroEL and
GroES and for folding is off set by enhanced generation of ATP by active TCA cycle due do increased active
aconitase. Above results are also substantiated by calculation of GroEL: aconitase ratio using relative band
intensities by gel documentation. An increase of 25%
in the GroEL: aconitase ratio was observed at 37 C as
compared to that at 25 C in the cells over expressing
aconitase and GroEL.
Time of incubation required after induction to over
expressed aconitase, for the pAco containing strain, in
presence of chaperonin GroEL only, was found to be
the least. Whereas, in presence of the complete folding machinery viz. GroEL and GroES the time required
was the maximum as compared to the over expression of
aconitase in absence of chaperonin. Earlier reports (Chen
& Mikecz, 2005; Song et al., 2003; Squier, 2001) reveal
that aggregate formation in cell causes reduction in cell
growth, inhibition of transcription and loss in cellular
functions. Over production of aconitase only may lead
to formation of insoluble aggregates in the cell, resulting
in reduced cell efficiency. GroEL has the property to trap
non-native aconitase, and preventing its aggregation in
cell enhancing efficiency of cell function, requiring less
time, of incubation after induction. In presence of exogenous GroEL and GroES, a large portion of energy in the
form of ATP is used up for correct folding, as chaperonin assisted protein folding is ATP driven reaction

1983

(Chaudhuri et al., 2001; Farr et al., 2003; Richardson

et al., 1998). This reduces the rate of protein synthesis (another ATP driven reaction) significantly. At lower
temperature the protein synthesis itself is very slow,
requiring larger time for complete induction to occur.
For complete induction of the Lacoperon system a
critical concentration of inducer is required to inactivate
the repressor protein produced by lacI gene. Addition of
IPTG binds to the active LacI repressor and causes dissociation from its operator. Use of IPTG as the inducer
for production of heterologous proteins has already been
tested (Schumann & Ferreira, 2004). The optimum concentration of inducer for aconitase expression was found
to be 75 M. At lower concentration of IPTG the amount
of aconitase produced was much less. Thus, varying concentrations of the inducer can be efficiently used as a
simple tool for controlling the expression of recombinant aconitase in E. coli.
Aconitase expression in E. coli seems to be affected
by the over expression of GroEL and GroES. In presence of GroEL alone, the amount of over expressed
aconitase reduces drastically. The reason may be that
the cells are giving a priority to the synthesis and folding of the helper protein GroEL so that less energy is
available for the synthesis of aconitase. In presence of
both the chaperonin, the reduction in aconitase expression is almost negligible as the presence of the complete
folding machinery increases the extent of correct folding
of newly synthesized aconitase. The excess ATP generated from enhanced TCA cycle through the participation
of increased amount of correctly folded aconitase may
serve two purposes: (a) enhancing cell growth rate and
(b) increasing rate of protein synthesis. The increase in
protein synthesis can be seen in the form of enhanced
aconitase expression in presence of both GroEL and
GroES.
Reduced expression of aconitase in the E. coli cells
over producing GroEL also supports the observation that
the least time of induction is required by cells overexpressing aconitase and GroEL simultaneously. Due to
high expression of aconitase in strains expressing the
entire chaperonin machineryGroEL and GroES, the
time of induction was very high. A large portion of the
cellular energy is involved in the folding process, resulting in a slower rate of protein synthesis and hence, a
higher time of induction.
Enhanced efficiency of correct folding of aconitase
in E. coli cells and the maximum percentage of native
aconitase was found in the case when both GroEL and
GroES chaperonin were co-expressed along with aconitase at 25 C. Even though, the rate of protein synthesis is
much higher at 37 C as compared to 25 C, the extent of

1984

P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 19751985

folding is higher at 25 C. The higher rate of aggregation

reaction at 37 C competes with the chaperone assisted
folding reaction, resulting in the loss of a large portion
of the newly synthesized protein as aggregated mass.
Generally, the extent of aggregation is greater at higher
temperature due to the strong temperature-dependence
of the hydrophobic interactions, which dominate protein aggregation (Miot & Betton, 2004). The exception being aconitase over expression in M15 cells at
37 C. Enhanced folding of aconitase is observed due
to increased expression of endogenous GroEL at higher
temperature. Small amounts of aconitase in the supernatant of the M15 cells over expressing recombinant
aconitase and GroEL were found. This is due to the
binding of the over expressed aconitase with GroEL cavity, without release of a folding competent intermediate.
The protein storage function of GroEL has been studied and reported (Llorca et al., 1998). When the cells
are disrupted and the protein denatured for loading in
the SDS-PAGE, the GroEL bound aconitase is released
and appears in the soluble fraction along with GroEL.
These results have been substantiated by aconitase assay
of all three strains at both the temperatures. Thus, the
aconitase that appears in the supernatant of M15 cells
over expressing recombinant aconitase in the presence
of both GroEL and GroES was found to be biologically
active.
Acknowledgements
The authors acknowledge the generous gifts of pAco
plasmid from Prof. Sabine Rospert and pGroEL and
pGroELS from Prof. A.L. Horwich. The work has
been supported by Ministry of Human Resource and
Development (MHRD), Govt. of India and Industrial
Research and Development Division (IRD), IIT, Delhi.
Ms. Nishtha Aggarwal and Ms. Pragya Batra are SURA
awardees from IRD, IIT, Delhi.
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