Bioresource Technology 123 (2012) 135–143
Contents lists available at SciVerse ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Production of soluble recombinant proteins in Escherichia coli: Effects
of process conditions and chaperone co-expression on cell growth and production
of xylanase
Kamna Jhamb, Debendra K. Sahoo ⇑
CSIR – Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India
h i g h l i g h t s
" Low temperature or reduced metabolism favored soluble protein expression.
" Co-expression of molecular chaperones resulted in 33–40% soluble Xyn-B expression.
" Soluble expression of Xyn-B was reproducible in a scalable semi-synthetic medium.
" High E. coli cell O.D. and growth rate could be due to inclusion body formation.
a r t i c l e
i n f o
Article history:
Received 18 May 2012
Received in revised form 6 July 2012
Accepted 7 July 2012
Available online 16 July 2012
Keywords:
Escherichia coli
Inclusion bodies
Soluble expression
Molecular chaperones
Xylanase
a b s t r a c t
In this study, effects of temperature, inducer concentration, time of induction and co-expression of
molecular chaperones (GroEL–GroES and DnaKJE), on cell growth and solubilization of model protein,
xylanases, were investigated. The yield of soluble xylanases increased with decreasing cultivation temperature and inducer level. In addition, co-expression of DnaKJE chaperone resulted in increased soluble
xylanases though the time of induction of chaperone and target protein had a bearing on this yield. A
combination of chaperone co-expression and partial induction resulted in 40% (in DnaKJE) and 33%
(in GroEL–GroES) of total xylanase yield in soluble fraction. However, the conditions for maximum yield
of soluble r-XynB and maximum % soluble expression of r-XynB were different. Higher expression of soluble xylanases in a scalable semi-synthetic medium showed potential of the process for soluble enzyme
production.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The extensive employment in recombinant protein production
and plethora of knowledge available on genetics and physiology
of gram-negative Escherichia coli has made it to be called as the
‘‘microbial factory’’. The ease of handling, inexpensive growth
requirements and the accumulation of the product to higher levels
in the cell cytoplasm are other features of this organism which
have aided in making it the most sought after expression host.
However, not all genes are expressed in E. coli in a facile manner.
There are several limitations associated with the production of
non-native/heterologous proteins in the prokaryotic system of
E. coli. It poses significant problems in post-translational modifications of proteins, having no capacity to bring about glycosylation or
disulfide bond formation. As a result, recombinant polypeptides
⇑ Corresponding author. Tel.: +91 172 6665324; fax: +91 172 2690632.
E-mail address: [email protected] (D.K. Sahoo).
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.07.011
are found to be sequestered within large refractile aggregates of
inactive protein known as inclusion bodies (IBs) (Georgiou and
Valax, 1996). Recovery of biologically active products from aggregated state is typically accomplished by unfolding with chaotropic
agents followed by dilution/dialysis into optimized refolding buffers. Optimization of the refolding procedure however, requires
time consuming efforts and is not conducive to high product yields
(Sorensen and Mortensen, 2005). Thus, maximizing the yields of
recombinant proteins in a soluble and active form in vivo becomes
an alternative to in vitro folding.
From an industry perspective, low product yields and high
recovery costs of a protein/enzyme from a microbiological source
is not acceptable and hence conditions have to be explored which
balance heterologous protein production and host physiology to
maximize soluble product yields. A number of approaches for the
redirection of proteins from IBs into the soluble fraction are described in the literature. Modification of cultivation conditions to
changing a host cell, or use of fusion partners are some of the
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K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
methods. Molecular chaperones and other folding catalysts have
long been known to aid the protein-folding pathway. Manipulation
of these folding modulators to increase the refolding efficiencies
in vitro in combination with immobilization technology has lately
been explored (Jhamb et al., 2008). The effects of co-expression
of molecular chaperones in vivo in increasing the solubility of client
proteins have also been well documented (Nishihara et al., 1998;
Martínez-Alonso et al., 2010; Sonoda et al., 2010; Cui et al., 2011;
Wang et al., 2011; Zelena et al., 2012). However, the success of
chaperone co-overproduction depends on the relative affinities of
the chaperone system to the folding intermediates and the folding
and aggregation kinetics of such species. These parameters are difficult to predict and as such, the application of molecular chaperones rely on trial and error experiments (Schlieker et al., 2002).
Another fact that needs consideration (de Marco and de Marco,
2004) is that the suitability of any chaperone combination is target
protein specific. Hence, fine tuning of the expression of both the
target protein and chaperones is required in order to improve the
amount of soluble protein.
Xylanases have gained immense interest in the past decade owing to their importance in the pulp and bleaching industries and
particularly as a biobleaching agent. Lack of hyper producing
microorganisms, production of complexed xylanolytic enzymes,
presence of cellulase contamination with xylanases that poses a
problem in paper industry (Subramaniyan and Prema, 2002) and
advent of recombinant DNA technology led to taming of xylanase
genes in homologous as well as heterologous systems such as
E. coli and Bacilli (Kulkarni et al., 1999; Beg et al., 2001; Pereira
et al., 2003; Ko et al., 2009). In the present study, xylanase from
Xanthomonas oryzae pv. oryzae (xynB) wherein a cellulose-binding
domain is absent (Rajeshwari et al., 2005) was taken as a model
protein for expression in E. coli. Absence of post-translational modifications such as glycosylation in E. coli and accumulation of recombinant xylanases in intracellular aggregates posses a major
limitation to their production for an industrial process (US Patent
6083734 – Chuang et al., 2000) and also limit their activity. As
the production of this important enzyme in soluble form is desirable, strategies were explored to avoid the formation of xylanase
inclusion bodies. The effect of E. coli cell’s process/growth conditions and co-expression of chaperones on production of xylanases
in soluble form were investigated.
2. Methods
2.1. Bacterial strains and plasmids
The gene encoding the secreted xylanase precursor, xynB
(978 bp) (obtained from Dr. K.V. Radha Kishan, IMTECH, Chandigarh), cloned in pGEX-2T vector (Amersham Biosciences) within
the restriction sites BamHI and EcoRI, under the control of tac promoter (inducible with IPTG) has been used in this study. It possesses an N-terminal GST-tag. The chaperone plasmids pGro7
and pKJE7 used for co-transformation with pGEX-2T xynB were obtained from Takara Bio Inc., Japan. These plasmids carry an origin
of replication derived from pACYC and a Chloramphenicol (CmR)
resistance gene. This arrangement allows their use with the ColE1
based plasmids used in E. coli expression systems. The chaperone
plasmids used with and without the xylanase encoding plasmid
are listed in Table 1. The plasmids were transformed individually
in BL21 cells which were then made competent using the CaCl2
method (Sambrook and Russell, 2001). The plasmid pGEX-2T xynB
carrying the gene for expression of the r-XynB protein was transformed into the competent cells carrying the plasmids pGro7 and
pKJE7, thus resulting in two strains expressing Gro7-XynB and
KJE7-XynB. The chaperone proteins are expressed under the control of araB promoter and can be induced with L-arabinose.
2.2. E. coli cells expressing r-XynB with and without folding accessory
proteins
E. coli BL21 expressing plasmid pGEX-2T-xynB with and without chaperone plasmid were grown overnight in 10 mL Luria Broth
(Difco, 10 g tryptone; 5 g yeast extract; 10 g NaCl per liter) containing the required antibiotics (100 lg/mL ampicillin ± 30 lg/mL
chloramphenicol) at 37 °C, 200 rpm following inoculation from a
single isolated colony obtained from the glycerol stock. This preinoculum was used at 1% v/v level to inoculate a 250 mL flask containing 50 mL Luria broth and the necessary antibiotics and the
flask was incubated at 37 °C, 200 rpm in an orbital shaker. In order
to study the growth and protein expression profiles, 1 mL samples
were taken at every 2 h interval. Induction of chaperones was done
with 0.5 mg/mL L-arabinose (Sigma, St. Louis, MO) after 2 h of inoculation while that of xylanase was done with 1 mM IPTG (Calbiochem) when the O.D.600nm reached 1.0. All samples taken were
processed for determination of cell biomass (O.D.600nm) and quantification of protein expression (per O.D.600 of the cells) by SDS–
PAGE (Laemmli, 1970). A control culture without induction was
also run in parallel.
2.3. Effect of process conditions on solubility of r-XynB
2.3.1. Inducer concentration
Two hundred and fifty milliliters flasks containing 50 mL LB
medium supplemented with ampicillin were inoculated at 1% v/v
level with pre-inoculum and incubated at 37 °C and 200 rpm. In
the mid-exponential phase (O.D.600nm = 0.8), the cultures were
induced with IPTG concentrations of 0.01, 0.1 and 1.0 mM each
and further incubated overnight. The cultures grown in two sets
were harvested at two stages, one set at 1 h post induction and
the other after overnight growth by centrifugation for 10 min at
6000 rpm (3864 g) and 4 °C (Sigma 6K15). The cell pellets resuspended in PBS buffer and kept on ice bath were subjected to Sonication (MISONIX S-4000 Sonicator) for 8 min at 26% amplitude
with 30 s on and 30 s off sequence. The lysates were centrifuged
for 15 min at 10,000 rpm (10733 g) and 4 °C and the supernatants
were preserved for xylanase activity assay and protein analysis.
The insoluble inclusion body pellets were resuspended in 50 mM
Tris, pH 8.0 and the sample was used for SDS–PAGE analysis. Total
r-XynB protein concentration (per cell O.D.), protein in insoluble (I)
and soluble (S) fractions (per mL of culture) were analyzed by SDS–
PAGE analysis and quantified by densitometry. Percent (%) solubility of the target protein was calculated as: [S/(I + S)] 100.
2.3.2. Low temperature
In order to study the effect of temperature on protein expression, the cultures grown at 37 °C and induced with two concentrations of IPTG i.e. 0.01 mM and 1.0 mM, were shifted to 20 °C at
different points after induction such as, 1 h post induction and
3 h post induction. In addition, for induction at 1.0 mM concentration, one flask was shifted 0.5 h prior to induction. In each case, the
cells were harvested in two stages, 1 h post temperature shift to
20 °C and after overnight growth. The procedures for harvesting
and analyses were same as described before.
2.4. Combined effect of chaperone co-expression and varying process
conditions
2.4.1. Inducer concentration of protein (IPTG) and low temperature
Two hundred and fifty millilters flasks containing 50 mL LB
medium supplemented with ampicillin (100 lg/mL) and chloramphenicol (30 lg/mL) were inoculated with pre-inoculum at
1% v/v level and incubated at 37 °C, 200 rpm. After 2 h of
growth, L-arabinose (inducer for the chaperones) was added at
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Table 1
Cell growth rates of various strains used under induced and un-induced culture conditions. Regression coefficients are mentioned in brackets.
Escherichia coli strain characteristics
a
b
c
Specific growth rate (l) (h
1
)
Strain
Plasmid (Selection marker)
Before induction (0–3 h)
After induction (3–7 h)
BL21 wild type
XynB control
XynB induced
Gro7 control
Gro7 induced
KJE7 control
KJE7 induced
Gro7-XynB control
Gro7-XynB induced
KJE7-XynB control
KJE7-XynB induced
None
pGEX-2T-xynB(Amp)b
pGEX-2T-xynB(Amp)
pGro7 (Cm)c
pGro7 (Cm)
pKJE7 (Cm)
pKJE7 (Cm)
pGro7 (Cm) + pGEX-2T-xynB(Amp)
pGro7 (Cm) + pGEX-2T-xynB(Amp)
pKJE7 (Cm) + pGEX-2T-xynB(Amp)
pKJE7 (Cm) + pGEX-2T-xynB(Amp)
1.002(0.99)a
1.034(0.93)
0.931(0.98)
0.982(0.99)
0.977(0.99)
1.026(0.99)
0.994(1.0)
1.074(0.98)
0.919(0.99)
1.073(0.98)
0.957(0.99)
0.340(0.97)
0.360(0.97)
0.485(0.97)
0.323(0.88)
0.508(0.90)
0.323(0.50)
0.195(0.72)
0.274(0.94)
0.623(0.95)
0.137(0.94)
0.057(0.81)
Regression coefficients.
Amp – Ampicillin.
Cm – Chloramphenicol.
a final concentration of 0.5 mg/mL, followed by induction with
varying concentration of IPTG (0.01, 0.1 and 1.0 mM) at
O.D.600nm = 1.0. For expression of cultures at 20 °C, the cultures
were shifted to the lower temperature right after induction and
harvested after 6 h of growth. The cell free samples were kept
for total protein analysis. The cell pellets were processed and
analyses were same as described before.
2.4.2. Effect of L-arabinose concentration on solubility and yield of
r-XynB
The effects of L-arabinose concentration were investigated by
varying L-arabinose concentrations i.e., 0.5, 2.0 and 4.0 mg/mL.
Each of these concentrations was tested in conjunction with three
IPTG concentrations i.e. 0.01, 0.1 and 1.0 mM.
2.4.3. Medium composition, and time and sequence of induction
Experiments were carried out with Gro7-XynB and KJE7-XynB
wherein, three phases of chaperone induction were used to study
their effect on the amount of active xylanase protein expression.
In the first case designated as ‘I’, induction with L-arabinose
(0.5 mg/mL) was done 1 h prior to IPTG induction. In the second
case, ‘Sim’, Xylanase and chaperones were simultaneously induced,
while in the third case ‘Post’, chaperones were induced 1 h later
than r-XynB. In each case, the effects of two IPTG concentrations
on protein expression were studied i.e. 0.01 and 1.0 mM. Cultures
were harvested after 6 h post induction, sonicated, centrifuged and
the supernatants were analyzed for enzyme activity by the DNS
method as described later. This study was carried out using two
different media: Luria Broth (Difco) and semi-synthetic medium
that contained (a) 8.7 g/L KH2PO4, (b) 6.0 g/L (NH4)2HPO4, (c)
1.0 g/L glucose, (d) 2.0 g/L yeast extract, 8.0 g/L tryptone, (e)
0.4 g/L MgSO4, and (f) 25 mL/L trace metal solution. Each component of the medium (a–f) was separately autoclaved at 121 °C for
20 min and complete medium was reconstituted by mixing the
sterile components aseptically.
2.5. Analytical procedures
2.5.1. Determination of specific growth rate for recombinant E. coli
cells
The specific growth rate constant, l was calculated from the
slope of the line obtained by plotting natural log of cell concentration (Ln O.D.600) against time (h) according to the equation
y = mx + c, where y is the Ln (O.D.), m is the slope, x is time (h)
and c is the intercept.
2.5.2. SDS–PAGE
Expression of target protein was analyzed using polyacrylamide
gel electrophoresis according to the method of Laemmli (1970).
12% resolving gels were used for studying expression of cultures
carrying xynB and xynB + pKJE7, while 8% gel was prepared for
the strain carrying xynB + pGro7. Staining of the gels was done by
Coomassie Blue R250 (Sigma, St Louis, MO). After staining and
destaining, the gels were analyzed using densitometry. A gel documentation unit (Syngene, UK) was used for estimating the relative
quantities of protein present in various bands observed on the gels
by employing the GeneTools software (version 3.00) of the system.
Relative intensity of the desired band was quantified by measuring
and on comparing its intensity with that of known concentration of
BSA protein standard loaded in the same gel.
2.5.3. Assay of xylanase activity
The xylanase activity was measured by determining the amount
of reducing sugar released from oat spelt xylan (Sigma Co, USA).
The reaction mixture consisted of 1 mL of 1% w/v xylan solution
and 1 mL appropriately diluted enzyme (supernatants after sonication of the cell broth) solution in 0.05 M sodium acetate buffer (pH
5.3). After incubation at 50 °C for 30 min, the reaction was stopped
by adding 3 mL DNS (3,5-dinitrosalicylic acid) reagent and the
solution was boiled for 5 min followed by addition of 2 mL distilled
water. The absorbance was measured at 540 nm in a UV–Visible
spectrophotometer against buffer as blank and the concentration
of xylose released was determined from the standard plot of xylose. Xylanase activity was expressed as micromoles of reducing
sugar (xylose) released per minute per milliliter of the enzyme
solution under the conditions described (Miller, 1959).
2.6. Microscopy
2.6.1. Phase contrast microscopy
E. coli cells from the various samples were resuspended in PBS. A
few drops of the cell suspension taken on a microscope slide were
observed and photographed using a microscope (Olympus BX51
microscope) fitted with a DP70 automatic camera system. A
phase-contrast objective (UPlan Fluorite; magnification, 100;
numerical aperture, 1.3) was used for phase-contrast microscopy.
2.6.2. Scanning electron microscopy (SEM)
E. coli cells were grown in Luria Broth to an exponential phase,
harvested by centrifugation and resuspended in 0.22 lm filtersterilized PBS. Pellets were washed thrice in PBS and each sample
was spread on a poly (L-lysine)-coated glass slides (18 18 mm) to
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K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
immobilize bacterial cells. Glass slides were incubated at 30 °C for
90 min. Slide-immobilized cells were fixed with modified Karnovsky’s fixative 1965 (David et al., 1973) and dehydrated with a
graded ethanol series. After freeze drying and platinum coating,
the samples were observed with a scanning electron microscope
(Zeiss EVO 40). Prints of negatives of scanning electron micrographs were made and overlaid with transparencies. Cell outlines
were traced, cut out, and weighed. The numbers of cells were
counted manually by point counting. The area of each cell was
computed by comparison with the weight of an area corresponding
to 100 lm2 cut out from the same transparency sheet. To calculate
the cell size, 41 (minimum) to 109 (maximum) individual cells
were counted for each test and control samples. Comparisons between control and test samples were done by two-tailed t-test
(Ramandeep et al., 2001).
3. Results and discussion
The model protein Xylanase (XynB) expressed in E. coli BL21
cells carrying plasmid pGEX-2T-xynB and possessing an N-terminal GST-tag was being sequestered in inclusion bodies as was evident from the phase contrast microscopy (data not shown) where
distinct inclusion bodies were visible after induction. It could also
be observed that the size and number of inclusion bodies varied
with the inducer concentration and phase of growth. When induced by IPTG, XynB protein accumulated to nearly 30% of the total
cell protein concentration, however, only 4% of expressed xylanase was in soluble form. Thus, various strategies including coexpression of chaperones were evaluated so as to direct the insoluble aggregates of xylanase into soluble form and the effect of
chaperone co-expression on cell growth and protein solubility
were investigated.
3.1. Cell growth and protein expression by E. coli
The growth profiles of E. coli BL21, both wildtype and cells containing plasmid pGEX-2T-xynB, with/without protein expression
were studied. It is known that the expression of recombinant proteins induces a stress response and hence it results in impaired
growth rates and lower increase in cell biomass, generally the cell
growth being inversely related to the rate of recombinant protein
synthesis (Hoffmann and Rinas, 2004). In the present study, the
presence of the plasmid did not cause any change in the growth
pattern of BL21 cells. However, after induction of XynB gene, there
was a 42.6% increase in specific growth rate of BL21 cells expressing XynB over that of the wildtype BL21 cells and higher specific
growth rates also led to increased inclusion body formation of
XynB. In a recent report, Iafolla et al. (2008) have also reported that
the amount of inclusion body formation is a strong function of cellular growth rate wherein at the fastest growth rate, nearly five
times EGFP are present in inclusion bodies.
One of the frequent observations during the recombinant protein expression in E. coli is that of the filamentation of cells (Jeong
and Lee, 2003). In present case, the growth rates of uninduced and
to be induced cells were similar till induction, however after induction, induced cells grew faster by 35%, the final O.D. of control cells
being 3.4 as compared to that of 5.5 in case of cells expressing rXynB. However, the expression of foreign protein in a host is
known to reduce its growth rate (Glick, 1995). Hence, microscopy
was sought to answer the reason for this discrepancy. Scanning
electron microscopy of E. coli BL21 cells (without plasmid), uninduced BL21 cells carrying pGEX-2T-xynB and the same after induction with 1.0 mM IPTG was carried out. On calculating the mean
cell size (lm2) of these samples, it was found that the mean size
of BL21 cells and uninduced BL21 cells containing XynB gene
was almost the same while that of induced cells expressing XynB
had almost doubled. This could be the reason for the increase in
O.D.600 of the samples containing E. coli BL21 cells expressing
XynB.
Fig. 1 shows the profiles of r-XynB protein expression, cell
growth and % solubility observed at the varying times of harvest.
The r-XynB production was observed to increase till 4 h postinduction after which the production was almost constant at
215.3 mg r-XynB/L at the end of fermentation. This was evident
from Fig. 1 (inset) which shows the gel depicting the r-XynB
protein as distinct band at 58 kDa, quantified with respect to
protein per equivalent O.D.600 of the cells. Majority of the protein
was being produced as insoluble aggregates as it was found that
only 5.0% of the xylanase protein was in the soluble fraction
when harvested after overnight fermentation. The solubility profile
of this protein did not show appreciable change during the entire
Fig. 1. Profiles of xylanase expression in E. coli BL21 carrying pGEX-2T-xynB, induced with 1 mM IPTG. The cells were grown at 37 °C and 200 rpm. Inset shows the SDS–PAGE
analysis of XynB expression.
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K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
3.2. Effect of process conditions on expression of soluble r-XynB
inside the cells resulting in higher light scattering. Lowering the inducer concentration from 1.0 to 0.01 mM had an effect on total
protein expression, with r-XynB concentration reducing by 9-fold
and 5-fold when harvested at 1 h and overnight post induction,
respectively on induction with 0.01 mM IPTG. However, % solubility of expressed XynB at 1 h post induction was increased from
1.6% with 1 mM IPTG induction to 12.7% with 0.01 mM IPTG induction. This apparent increase in soluble protein could be accounted
for by overall low accumulation of expressed target protein with
induction at 0.01 mM IPTG concentration as the r-XynB was
expressed at a very low level and there by avoided crowding effect.
Partial induction has also been reported to increase protein solubility in vivo and a decrease in inclusion body aggregates of CorA protein was reported when the same were induced by low inducer
concentration (Chen et al., 2003).
3.2.1. Inducer concentration
Fig. 2 summarizes the studies on the effect of inducer concentration on r-XynB solubility which also showed no effect of IPTG
on specific growth rate. However, there was a significant difference
in the growth profiles with final O.D.600nm varying by a factor of 2
when cultures were induced by 0.01 mM IPTG or 1.0 mM IPTG. This
was probably due to enlarged cells with inclusion body formation
3.2.2. Low temperature
Inductions at IPTG concentration of 0.01 and 1.0 mM were further manipulated by lowering the growth temperature in order to
augment the solubility of the desired active protein. A combined
effect of low temperature and inducer concentration was studied
by shifting the cultures, induced with 0.01 and 1.0 mM IPTG
to 20 °C at varying time points in the course of fermentation as
cultivation period. Though, initially a small fraction of total protein
went into the soluble fraction (at 0.5 h post-induction), thereafter
the insoluble fraction of the enzyme increased. Maximum soluble
enzyme protein of 1.36 mg/L was observed at overnight (22 h)
post-induction.
It is well known that the overproduction of heterologous protein imposes metabolic burden on the host cell. The inclusion body
production occurs when the in-house capacity of the cell to fold
the protein intermediates is substantially overwhelmed and is no
longer able to prevent the deposition of protein folding intermediates into aggregates. In the present study, various strategies were
used, including process and molecular, to improve the solubility
of XynB, which otherwise would have formed inclusion bodies.
Pre
0.01
0.1
1.0
Pre
0.01
0.1
1.0
O.D(600nm)
8
1h
6
r-XynB
4
2
0
0
2
Uninduced
3
4
Time(h)
0.01mM
6
22
0.1mM
Overnight
r-XynB
1.0mM
B.
A.
250
14
12
10
150
8
6
100
4
% Soluble Xylanase
Total r-XynB(mg/L)
200
50
2
0
C.
0
0.01mM/1h
0.1mM/1h
1.0mM/1h
0.01mM/Ot
0.1mM/Ot
1mM/Ot
Inducer Concentration/Time of Harvest
r-XynB(mg/L)
% Solubility
Fig. 2. Effect of inducer (IPTG) concentration on production and solubility of r-XynB when grown at 37 °C and 200 rpm, and harvested at 1 h post induction/after overnight
growth. Inset A: growth profile of E. coli BL21 carrying pGEX-2T-xynB and induced by various IPTG concentrations. Inset B: SDS–PAGE analysis of expressed proteins at
different IPTG concentrations and harvested at 1 h post induction or after overnight growth (Ot). Inset C: profiles of total and soluble xylanase expression when induced by
varying IPTG concentrations i.e. 0.01, 0.1 and 1.0 mM. The cultures were harvested for analysis at 1 h post induction after overnight (Ot) growth.
Table 2
Cell growth rates and total r-XynB expression in E. coli at different culture conditions.
Condition
Control (37 °C)
Shift 0.5 h pre induction
Shift 1 h post induction
Shift 3 h post induction
Specific growth rate (l)
XynB(mg/L)
Before induction
Post induction (1 h shift)
Post 3 h shift
4h
Overnight
0.326(0.91)
0.168(0.90)
0.321(0.91)
0.337(0.92)
0.201(0.99)
0.106(0.99)
0.104(0.96)
0.193(0.98)
0.449(0.98)
0.113(0.99)
0.080(0.99)
0.052(0.85)
60.75
13.66
25.66
43.96
152.32
89.19
82.66
129.89
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K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
mentioned earlier (Table 2). When induced with 1.0 mM IPTG and
shifting the cultures post-induction to lower temperature, a decline in specific growth rate and expression of target protein was
observed. On the other hand, no effect on cell growth was observed
under similar conditions when induction was carried out with
0.01 mM IPTG.
Lower growth temperatures often lead to reduced inclusion
body formation owing to slower rate of protein synthesis, changes
in the level of cell stress, the decreased rate of aggregation and decreased hydrophobic interactions between nascent polypeptides
(Fink, 1998). However, in present case, the lower temperature
alone could not lead to an increase in r-XynB solubility (Fig. 3).
Inducing with 1.0 mM IPTG and shifting to 20 °C at varying points
of post-induction time did not increase % soluble xylanase, yet,
when the culture was shifted to 20 °C at 1 h post-induction and
incubated overnight (IIO), there was an increase in the soluble enzyme activity by 5-fold, as the quantity of XynB protein in soluble
fraction was found to be more than that in the control flask. Compared to 1.0 mM IPTG, % solubility of the r-XynB was higher with
0.01 mM IPTG as observed earlier. The maximum % solubility of
r-XynB achieved was 16.6% when the culture was shifted 1 h post
induction and harvested 4 h later (II4). When the same culture was
harvested overnight, there was decline in % solubility in r-XynB,
but the soluble enzyme activity increased 7-fold due to the increase in expression of total r-XynB. This indicated that either by
partially reducing the heterologous gene by using low inducer concentration or by reducing the cell growth (metabolism) of xylanase
producing E. coli cells by lowering the growth temperature and
allowing the protein to accumulate slowly over a longer period
of time might result in correctly folded protein. Similar observation
Fig. 3. Profiles of solubility of xylanase protein (after induction with 0.01 mM/1.0 mM IPTG) obtained at lower growth temperature of 20 °C and varying post induction time
points. C – Control at 37 °C, II – Shift to 20 °C 1 h post induction, III – Shift to 20 °C 3 h post induction. 1,4,O - Harvest after 1 h, 4 h and overnight after shift.
Fig. 4. Growth profiles of E. coli BL21 (wildtype) and E. coli BL21 cells carrying pGEX-2T-xynB and chaperone plasmids and with/without induction. All cells were grown at
37 °C and 200 rpm.
K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
of accumulation of soluble active protein in E. coli was made in case
of b-1,4-xylanase cloned from Saccharophagus degradans 2-40 at
low growth rate and induction at low IPTG concentration (Ko
et al., 2009).
3.3. Effect of co-expression of chaperones
One reason for aggregation of over-expressed proteins is the
insufficient level of chaperones to stabilize the misfolded conformers. Chaperones GroEL, GroES and DnaK, DnaJ and GrpE are folding
catalysts that act via preventing aggregation of nascent chains and
unfolding of misfolded aggregates. A number of publications demonstrated that co-overproduction of GroEL/ES or DnaK increased
the solubility of recombinant proteins to various extents by promoting the de novo folding of the client protein (Nishihara et al.,
1998; Gupta et al., 2006; Alibolandi et al., 2010; Sonoda et al.,
2010; Wang et al., 2011; Zelena et al., 2012). It was therefore
tempting to use molecular chaperones to optimize the folding process of xylanase as a model protein. However, there is no universal
approach in using chaperones that can be applied to all cases.
Moreover, the folding mechanism of xylanase (r-XynB) is not
known and hence it was difficult to predict as to which chaperone
system might help in this protein’s folding. Chaperone sets developed by HSP Research Institute (Takara Inc.) i.e. pGro7 and pKJE7
were employed in this study. The strains so constructed and used
in this study are summarized in Table 1 along with the specific
growth rate constants (l) as measured with the various plasmid
combinations in E. coli BL21 cells with the cells induced and uninduced.
Most notably, changes in the physiology of the producer cells
are detected by changes in the specific growth rate, which often
seriously declines due to the impact of plasmid presence and
141
expression of plasmid-encoded recombinant genes on the host cell
(Hoffmann and Rinas, 2004). This was evident from investigations
when pGro7 and pKJE7 were transformed individually and as a
combination with pXynB in BL21 strain. Even when the cells were
uninduced a decrease in l was also observed, however, as in case
of pXynB, in Gro7-XynB an increase in specific growth rate
(83.2%) was observed after induction. SEM micrographs also
showed increased cell elongation (not shown). The negative effect
on growth rate was highly pronounced in KJE7-XynB carrying cells,
induced or uninduced (Fig. 4). The fact that this set of folding modulators has not been consistently successful in enhancing the solubility of target recombinant proteins and this enhancement of
solubility, often being achieved at expenses of protein yield (quantity), was previously attributed to cell growth inhibition and proteolysis of recombinant protein (Martínez-Alonso et al., 2010; Kim
et al., 2005).
3.4. Combined effect of chaperone co-expression and variations of
process conditions
3.4.1. Inducer (IPTG) concentration and low temperature
A combined effect of varying inducer concentration, chaperone
co-expression and lower temperature was studied on the expression and solubility of r-XynB and the results are summarized in
Fig. 5. Without co-expression of chaperones, lowering the IPTG
concentration to 0.01 mM IPTG at 37 °C led to 20% soluble XynB.
Lowering the inducer (IPTG) concentration however, did not have
an impact on the protein solubility when chaperones were co-expressed. With GroEL–ES co-expression, at 0.1 mM IPTG induction,
35% soluble xylanase was obtained which was 10.5-fold more
enzymatically active as compared to r-XynB expressed without
any chaperone at the same induction condition. When grown at
Fig. 5. (A) Profiles of total protein and % solubility (% S) of expressed protein in E. coli strains expressing XynB, Gro7-XynB and KJE7-XynB when grown at 37 °C and 200 rpm.
(B) SDS–PAGE analysis of r-XynB when produced in the strains Gro7-XynB and KJE7-XynB. Protein band numbers 1, 2, 3 correspond to GroEL, XynB, DnaK respectively. (C)
Profiles of total protein and % solubility (% S) of expressed protein in E. coli strains expressing XynB, Gro7-XynB and KJE7-XynB when grown at 20 °C and 200 rpm and induced
with varying concentrations of IPTG.
142
K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
Table 3
Percentage solubility and enzyme activity values of expressed xylanase in E. coli cells expressing Gro7-XynB and KJE7-XynB.
concentration
(mg/mL)
IPTG concentration
(mM)
Gro7-XynB
% Soluble
xylanase
Enzyme activity
(U/mL)
% Soluble
xylanase
Enzyme activity
(U/mL)
0.5
0.01
0.1
1.0
0.01
0.1
1.0
0.01
1.0
12.28
12.81
13.53
12.12
12.96
13.08
13.25
12.09
2.22
3.77
4.39
12.55
8.93
6.56
10.65
5.61
23.65
13.98
34.23
35.16
35.10
34.99
34.82
35.36
0.57
0.98
1.14
0.76
1.07
1.29
0.97
1.62
L-ara
2.0
4.0
KJE7-XynB
Molecular chaperones were induced by varying L-arabinose (0.5, 2.0 and 4.0 mg/mL) and xylanase was induced by varying IPTG (0.01, 0.1 and
1.0 mM) concentrations. All cells were grown at 37 °C, 200 rpm.
Table 4
Percentage solubility of expressed xylanase in E. coli expressing Gro7-XynB and KJE7-XynB.
Induction condition
0.5/0.01 (I)
0.5/0.01 (Sim)
0.5/0.01 (Post)
0.5/1.0 (I)
0.5/1.0 (Sim)
0.5/1.0 (Post)
Gro7-XynB (% Soluble xylanase)
KJE7-XynB (% Soluble xylanase)
LB
Semi-Syn
LB
Semi-Syn
4.23
13.25
33.52
8.22
6.97
15.21
16.95
22.46
23.61
15.14
14.08
10.45
2.28
3.96
43.10
10.30
8.91
25.15
38.71
35.89
22.32
17.17
18.30
13.28
Molecular chaperones were induced at different post xylanase induction time points. ‘I’ stands for induction of chaperones done 1 h
prior to xylanase induction. ‘Sim’ stands for induction of chaperones done simultaneously with xylanase induction. ‘Post’ stands for
induction of chaperones done 1 h post xylanase induction. Inducer concentrations used were 0.5 mg/mL L-arabinose for each of the
chaperones and 0.01 and 1.0 mM IPTG for xylanase. All cells were grown at 37 °C and 200 rpm in two different media i.e. Luria Broth
(LB) and Semi-Synthetic (Semi-Syn) medium.
20 °C, the total protein expression declined and so did the
chaperone quantity. Positive effect of lowering the temperature
was seen when the cells were induced with 1.0 mM IPTG, wherein
% of r-XynB expressed in soluble fraction and enzyme activity were
found to double. The variable effects of this chaperonin may be due
to the fact that the size of the target protein (xylanase, 58 kDa) is
on the extreme end of the preferred GroEL substrate size range
(10–60 kDa) (Ewalt et al., 1997; Houry et al., 1999). When co-expressed with DnaKJE at 37 °C, comparatively higher % solubility
was achieved at tested concentrations of IPTG, however the comparative rise in enzyme activity was not observed indicating a loss
in protein quality, similar to one reported by Garcia-Fruitos et al
(2007). The soluble expression of r-XynB did not increase when
fermentation was carried out at 20 °C except in case of induction
at 1.0 mM IPTG, wherein an increase in soluble expression of rXynB from 17% to 26% was observed.
3.4.2. Concentration of chaperone inducer
As shown in Table 3, the effect of L-arabinose (L-ara) as an inducer of the genes for the chaperones GroEL–ES on the soluble
expression of r-XynB was studied. The total enzyme activity was
found to be highest at 2.0 mg L-ara/mL and 0.01 mM IPTG i.e.
12.55 U/mL, with nearly 12% soluble protein.
When varied concentration of L-arabinose was used to optimize
the solubility of r-XynB with DnaKJE co-expression, it was found
that at 4.0 mg L-arabinose/mL and 1.0 mM IPTG concentration,
maximum protein solubility was observed (35.8%). Even at higher
concentrations of L-ara, DnaK protein was not able to increase the
r-XynB solubility and the maximum enzyme activity was only
1.63 U/mL.
3.4.3. Medium composition, sequence and time of induction
The effect of order of induction of chaperones and xylanase protein on the target protein’s solubility was studied in two media
namely, Luria Broth (LB) and semi-synthetic medium (SS). When
grown in LB medium, with either of the chaperone systems i.e.
DnaKJE or GroEL–ES the induction of chaperones were observed
to affect the production of xylanase as presented in Table 4. The
induction of chaperones 1 h prior to xylanase led to a lower total
enzyme expression than when xylanase was induced first.
GroEL–ES is known to bind to partially folded polypeptides in a
post-translational manner and directs their folding in a proper conformation (Schlieker et al., 2002). When XynB was induced with
0.01 mM IPTG, the partial induction of the gene coupled with supply of exogenous GroEL–ES 1 h post induction of XynB, led to 33%
soluble protein which was biologically active. In case of KJE chaperone machinery which folds the nascent polypeptide chains cotranslationally, nearly 40% of total expressed xylanase was obtained as soluble xylanase when induced with 0.01 mM IPTG and
chaperones induced at 1 h post induction of XynB. In the case when
cultures were grown in semi-synthetic medium, as compared to
that in LB medium a higher percentage of soluble xylanase was
found, however, insignificant effect of the timing of induction of
chaperones with respect to xylanase protein was found. The reason
could be attributed to the higher overall production of chaperone
proteins (GroEL and DnaK) in the semi-synthetic medium with respect to that in LB medium (data not shown) thus negating the effect, if any, of the order of induction of chaperones.
4. Conclusion
In this work, a production strategy based on co-expression of
chaperones, low process temperature and inducer concentration,
and variation in time of induction of target gene and chaperone
was used to reduce the aggregation of xylanase and increase its
expression as soluble protein and activity. The results suggested
that low temperature facilitated proper folding and solubility of
XynB protein and GroEL–GroES system better assisted in folding
K. Jhamb, D.K. Sahoo / Bioresource Technology 123 (2012) 135–143
and solubilization of XynB protein in E. coli as compared to DnaKJE
system of chaperones and up to 40% expression of xylanases in soluble fraction was obtained.
Acknowledgements
Ms. Kamna Jhamb gratefully acknowledges Council of Scientific
and Industrial Research, Government of India for her fellowship.
The help of Dr. Manoj Raje, IMTECH, Chandigarh for Scanning Electron Microscopy (SEM) is thankfully acknowledged.
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