Iranian Biomedical Journal 19 (2): 102-110 (April 2015)

DOI: 10.6091/ibj.1338.2015

Efficient Process Development of Recombinant Human

Granulocyte Colony-Stimulating Factor (rh-GCSF)
Production in Escherichia coli
Valiollah Babaeipour*1, Sirwan Khanchezar2, Mohammad Reza Mofid3
and Mahdi Pesaran Hagi Abbas4
1

Dept. of Bioscience and Biotechnology, Malek Ashtar University of Technology, P.O. Box 14395-1561, Tehran,
Iran; 2Dept. of Biotechnology, Chemical Engineering Faculty, Tarbiat Modarres University, Tehran, Iran;
3
Dept. of Biochemistry and Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Science,
Isfahan University of Medical Sciences, Isfahan, Iran; 4Dept. of Life Science Engineering, Faculty of New
Technologies, University of Tehran, Tehran, Iran
Received 5 April 2014; revised 27 August 2014; accepted 31 August 2014

ABSTRACT
Background: The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of
white blood cells and plays an important role in medical treatment of cancer patients. Methods: An efficient
process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate
was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific
growth rate was maintained at the maximum value of 0.55 h-1 at the beginning of feeding to 0.4 h-1 at the induction
time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were
released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation
exchange chromatography. Results: Obviouly, higher specific growth rate decreases process time and consequently
increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l-1 and 32.1 g.l-1.
Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g-1 DCW and 1.83 g.l1 -1
.h , respectively. According to the available data, this is one of the highest YP/X and productivity that has been
reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and
purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen
and PD-Grastim showed that all proteins have a similar secondary structure. Conclusion: Modified exponential
feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and
maximum productivity. Iran. Biomed. J. 19 (2): 102-110, 2015
Keywords: Escherichia coli, Granulocyte colony-stimulating factor (GCSF), Process development

INTRODUCTION

uman granulocyte colony-stimulating factor

(GCSF) is a single chain polypeptide
composed of 174 residues, (MW = 18.8 kDa).
GCSF is a hemopoietic growth factor that stimulates
proliferation, differentiation, and functional activation
of blood cells. Active GCSF contains a free cysteine at
position 17 and two intramolecular disulfide bonds
formed between positions 36/42 and 64/74 [1-3].
It is well known that E. coli is a useful host for
production of recombinant proteins [4-9], since it has
simple nutrient requirement, well known molecular
genetics and cellular physiology, and high growth rate
on inexpensive substrate [4, 5, 9]. In the case of

intracellular
recombinant
protein
expression,
productivity is proportional with the final cell density
and the specific yield (i.e. the amount of product
formed per time unit). Therefore, fed-batch cultivation,
which is an important method to achieve high celldensity culture, improves productivity of target
products. Suitable feeding strategies in fed-batch
cultivation regulate available nutrient concentration
and consequently affect specific growth rate, maximum
cell concentration, the specific yield of recombinant
protein, and formation of by-products [6, 10-12].
Exponential feeding is one of the most widely used
approaches that allow implementation of the process
and manipulation of specific growth rate [6, 12-14] by
control of limiting substrate. In the exponential feeding

*Corresponding Author; Tel.: (+98-21) 2297 4614; Fax: (+98-21) 2297 4604; E-mail: [email protected]

Iran. Biomed. J., April 2015

Efficient Process Development of rh-GCSF Production in E. coli

method, cells grow under constant specific growth rate

by increasing feeding rate corresponding to cell
concentration and inhibit acetate formation by
maintaining specific growth rate below the critical
value. Therefore, keeping at a maximum attainable
level and its control under a critical value before
induction provide the nutrients at suitable concentration ranges, which is very important for achieving
high cell density and productivity [15,16].
Overexpression of heterologous proteins in E. coli
cytoplasm often results in the formation of insoluble
aggregates, so called inclusion bodies. Production of
recombinant protein as inclusion body has a drawback
and some benefits. Inclusion body contains high degree
purity of target protein, and inactive proteins can be
protected from proteolysis [17]. Also, it can be
recovered from cell lysate (mechanically disrupted
cell) by simple centrifugation [18]. The main challenge
in recombinant protein production as inclusion body is
the isolation of active proteins from inclusion bodies.
This process includes inclusion body solubilization in
high concentration denaturant agents (such as urea or
guanidine hydrochloride) and protein refolding for
remove of denaturant in the presence of oxidizing
agent [19-21]. Generally, inclusion bodies are
solubilized at extreme pH that is special for each
protein. In the case of recombinant protein contain
disulfide bond, appropriate redox condition is needed
for protein refolding [17].
A few researches have shown that the recombinant
human GCSF (rh-GCSF) expression level in E. coli is
at moderate to high level [20,13], but the yield of final
product is poor. Hence, for the first time in this study,
we tried to obtain higher yield and productivity by
keeping specific growth rate at a maximum attainable
level during the exponential feeding of fed-batch
cultivation. We also investigated the effect of feeding
strategy on by-products and medium ingredient
concentration, plasmid stability, and total process time
in the fed-batch process during high cell-density
culture of recombinant E. coli BL21 (DE3) [pET23ahgcsf]. Then we tried to develop an efficient process
for purification of rh-GCSF. Purified rh-GCSF was
characterized with circular dichroism and size
exclusion chromatography rather than standard
samples, such as Neupogen (Roche, Germany) and
PD-Grastim (Pooyesh Darou, Iran).

MATERIALS AND METHODS

Microorganism and vector system. The host cell, E.
coli BL21 (DE3) (Novagen, United States), which
contained pET23a inducible expression vector
(Novagen, United States) carring the rh-GCSF gene
[21] at NotI and NdeI sites. Transformed cells using the

103

calcium chloride procedure were cultured on LuriaBertani (LB) agar plates containing 100 mg/l
ampicillin.
Media and solutions. Transformed E. coli was
cultivated on LB agar medium and M9 modified
medium, which were used as seed culture and
fermentation medium. M9 modified medium contained
10 g glucose, 15 g K2HPO4, 7.5 g KH2PO4, 2 g citric
acid, 2.5 g (NH4)2SO4, 2 g MgSO4.7H2O, and 1 ml
trace element solution per liter. The trace element
solution consisted of 2.8 g FeSO4.7H2O, 2 g
MnCl2.4H2O, 2.8 g CoSO4.7H2O, 1.5 g CaCl2.2H2O,
0.2 g CuCl2.2H2O and 0.3 g ZnSO4.7H2O per liter in 1
M HCl, and each medium contained 100 mg/l
ampicillin. Fed-batch cultivation was carried out in a 2
L bench-top bioreactor (Inforse AG, Switzerland) with
the working volume of 1 L, including two six-blade
Rushton impellers with a speed range of 50-1,200 rpm.
Analytical procedure. Cell growth was monitored by
measuring culture turbidity at 600 nm and dry cell
weight (DCW). For determination of DCW, pellet from
5 ml broth culture was dried at 105C until constant
weight [22]. Glucose, ammonia, phosphate, and acetate
were analyzed enzymatically by using the appropriate
kits (Chemenzyme, Iran and Boehringer Mannheim/RBiopharm, Germany). The expression of rh-GCSF was
analyzed by Coomassie brilliant blue-stained SDSPAGE with 12.5% polyacrylamide [23] and quantified
by a gel densitometer. Total soluble protein was
analyzed by Bradford's method [24]. After SDSPAGE, the gel was transferred to and blotted on the
PVDF membrane in order to recognize rh-GCSF [23,
25]. The plasmid stability was determined by plating
samples from fermentation broth on LB agar plates
with and without ampicillin. Then the fraction of
plasmid-containing cells on LB with ampicillin to
those on LB without the antibiotic were calculated as
plasmid stability [22].
Fed-batch cultivation. A batch culture was initially
established by the addition of 100 ml of an overnightincubated seed culture grown at 32oC and 100 rpm
(OD600 = 1.4-1.6). pH was maintained at seven by the
addition of 25% (w/v) NH4OH or 3M H3PO4 solution.
Dissolved oxygen was controlled at 30-40% (v/v) of
air saturation by controlling both inlet air (which was
enriched with pure oxygen) and agitation rate. Foam
was controlled by the addition of silicon-anti-foaming
reagent. After depletion of initial glucose in the
medium, as indicated by a rapid increase in the
dissolved oxygen concentration, the feeding was
initiated. Feeding rate was increased step by step based
on the exponential feeding strategy with maximum
attainable specific growth rate during fed-batch

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Babaeipour et al.

cultivation. The exponential feeding was determined

by the following equation (equation 1) [26]:
(1)

Where V0 is volume of the medium in the

bioreactor (l), X0 is biomass concentration at the start
of feeding g(DCW) l-1, t is the time (h), is the
specific growth rate (h-1), S0 is the glucose
concentration (g l-1) which is 400 g l-1 in the feeding
solution, F(t) is feeding rate (l h-1), M(t) is mass
feeding rate (g h-1), YX/S is the yield of biomass as a
result of substrate (g DCW g-1 glucose), t0 (h) is the
starting time for each feeding step, and m is the
specific maintenance coefficient (g g-1 h-1). Feeding
rate was corrected by the turbidity of taken samples
every 10 minutes.
The coefficient yield (YX/S) and maintenance
coefficient (m) were set at 0.5 and 0.025 g g-1 h-1,
respectively. For development of a simple feeding
strategy with the highest attainable specific growth rate
during the entire process, a maximum oxygen transfer
capacity was applied to the bioreactor, and glucose
concentration was maintained below 2 g l-1 by a
gradual increase in feeding at each step.
Cells were induced by the addition of 2 mM IPTG in
all experiments. The required nitrogen source
(ammonium) was supplied by the addition of 25%
(w/v) NH4OH, which was also used for maintaining pH
at 7. The temperature of the process was maintained at
37C, and the acetate and glucose concentrations were
controlled manually at 10-min intervals.
Purification of recombinant human-granulocyte
colony-stimulating factor. The fermented broth was
centrifuged at 8,000 g at 4C for 30 min, and the
obtained pellet was washed twice with 50 mM
phosphate buffer (pH 7.4). The wet cells (50 g per 200
ml lysis buffer) were suspended in lysis buffer,
containing 100 mM Tris-HCl, 1 mM EDTA and 1 mM
phenylmethylsulfonyl fluoride. Afterward, the cells
were broken by passing the suspension through a
homogenizer three times at 800 bar (Niro Soavi, North
America). The cell homogenate was centrifuged,
supernatant was discarded, and inclusion bodies were
recovered.
Next, inclusion body concentration was measured
using Bradford's method [24]. Then it (1 g inclusion
body per 5 ml) was resuspended in first washing buffer
(2.5 g l-1 Triton X-100, 50 mM Tris-HCl, 5 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride [pH 8],
and 0.01 g/ml DNase1) incubated for 40 min and
recovered by centrifugation at 8,000 g at 25C for 30
min. Second washing step, the inclusion body pellet,

Iran. Biomed. J., April 2015

was resuspended in second washing buffer (2 M urea)

and incubated for 40 min and then recovered at 8,000
g at 25C for 30 min. Then, 350 mg of washed
inclusion bodies was dissolved in 10 ml inclusion body
solubilization buffer (30 mM Tris-HCl [pH 12], 1 mM
EDTA, 8 M urea, and 100 mM reduced glutathione.
The solution was incubated at 25-28oC for 45 min and
spun down to remove insoluble cell debris. The rhGCSF was refolded by adding protein solution to
refolding buffer (3 mg protein per ml refolding buffer
with stirring), containing 30 mM Tris-HCl (pH 5), 3 M
urea, 20 mM glutathione, 2 mM oxidized glutathione,
and 1 mM EDTA). The pH of refolded protein solution
was adjusted to 5 with 2 M acetic acid. Subsequently,
refolded protein solution was applied in cation
exchanger, Tricorn mono S 10/100 GL column (GE,
USA), which was previously equilibrated with three
bed volume of buffer A (20 mM Na-acetate, pH 5.0) at
4 ml/min. A 50-ml linear gradient to 100% buffer B (1
M NaCl in buffer A) was applied at 2 ml/min. The
fractions containing the desired rh-GCSF were pooled
and concentrated using Amicon Ultra (Millipore, USA)
with a molecular mass cut-off of 5 kDa by
centrifugation at 4,500 g at 4 oC for 60 min.
Concentrated protein solution was applied to a
Superdex 75 (16/60) column (GE Healthcare, USA),
which had equilibrated with GFC buffer (20 mM
sodium acetate, pH 5.0). Isocratic elution was
performed with buffer A at 0.8 ml/min, and 2.4-ml
fractions were collected. Fractions containing rh-GCSF
were pooled and stored at -80C, and protein
concentration was determined based on the calculated
extinction coefficient of 15820 M-1cm-1 at 280 nm for
rh-GCSF.
Circular dichroism measurement. The purified rhGCSF alongside innovator product (Neupogen) was
analyzed with 10-M protein in 10 mM citrate buffer
(20.5 ml citric acid and 29.5 ml sodium citrate, pH 5)
at 22C by Jasco J-715 spectropolarimeter using 2 mm
path length cylindrical cell [27].
Analysis of monomer and aggregates. Size
exclusion chromatography was carried out by using
TSK-GEL G3000SWXL (300 mm 7.8 mm, Tosoh,
Japan) column chromatography system with
photodiode array detector. The mobile phase consisted
of K2HPO4-Na2HPO4 1 mM in water with pH of 6.2.
Flow rate was maintained at 0.6 ml/min, and analysis
was carried out at the wavelength of 280 nm [28].
Bioactivity assay. In this study, in vitro biological
activity assay of rh-GCSF was evaluated by
proliferation of HL-60 treated with DMSO. At first,
100 l of HL-60 cell suspension (2 105 cell/ml) was
added into a 96-well microplate. Then DMSO and RA

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Efficient Process Development of rh-GCSF Production in E. coli

(1.3% and 0.1 M, respectively) were added to the

each well and incubated at 37oC for 2 days in 5% CO2.
Different dilutions of rh-GCSF were added to each
well and incubated for two days at the same conditions.
The reversal rate of the cells from the differentiative to
the proliferative phase was determined by MTT assay.
Finally, cell proliferation was measured by reductase
mitochondrial enzyme through the reduction of MTT
to Formazan [3].
RESULTS
Due to metabolic burden of recombinant protein
overexpression on E. coli as well as decrease in
specific growth rate during induction [29], experiments
were designed to obtain a feeding rate, which would
lead to the higher attainable specific growth rate and
consequently, higher cell concentration before
induction. In this study, feeding rate was increased
stepwise by maintaining the glucose concentration
within a permissible range and using the maximum
oxygen transfer capacity of the bioreactor. Figure 1
shows feeding rate variation during fed-batch
cultivation of E. coli BL21(DE3) [pET23a-gcsf].
Maximum oxygen transfer rate was achieved by
increasing the impeller rotation speed from 400 rpm to
1,200 rpm during the process.
The exact equation for time variation in the specific
growth rate was correlated by using experimental data
obtained from various fed-batch cultures of E. coli
BL21 (DE3) [pET23a-hgcsf] under the glucose nonlimited conditions (equation 2):
(2)

Batch Stage

Fed-batch Stage

0.6

160
Induction

0.5

140
120

0.4

100

0.3

80
60

0.2

40
0.1

Dry cell weight (g l-1)

Fig. 1. Feeding rate variation during fed-batch cultivation of

E. coli BL21(DE3) [pET23a-gcsf].

105

t0 is time at the start of feeding, t is the time of process,

and R2 is coefficient of determination for equation 2.
Equations 1 and 2 were used to determine the feeding
rate in the fed-batch process. Figure 2 shows the results
of using this feeding strategy on fed-batch cultures of
un-induced recombinant E. coli BL21 (DE3) [pET23ahgcsf].
Figure 2 shows DCW and specific growth rate
change during both un-induced and induced fed-batch
culture of recombinant E. coli. Final cell concentration
of un-induced fed-batch culture after 17.5 0.5 was
145 5 g (DCW) l-1. Based on the results, cell density
in both fed-batch culture reached 75 g (DCW)l-1 during
the first 14 h, while specific growth rate was decreased
from 0.55 to 0.43 h-1. During feeding stage of fedbatch cultivation, the specific growth rate was
decreased from 0.55 to 0.04 h-1. Moreover, decrease in
specific growth rate of induced fed-batch culture was
more than un-induced one. Maximum cell density and
rh-GCSF concentration in induced process were 125
5 g(DCW) l-1 and 32 1 g(GCSF) l-1, respectively. In
addition, the final specific yield (YP/X) and productivity
of rh-GCSF in induced fed-batch culture were 250 10
mg(GCSF) g-1 (DCW) and 1.83 0.05 g (GCSF) l-1 h1
, respectively. Plasmid concentration in pre-induction
stage of fed-batch culture was maintained constant and
increased after induction (Fig. 3). As illustrated in
Figure 4, plasmid stability was higher than 95% entire
process. A slight decrease in plasmid stability after
induction was related to metabolic burden of
recombinant protein over-expression on host cells.

Specific growth rate (h-1)

Iran. Biomed. J., April 2015

20

0
0

10 12 14 16 18

Time (h)
Fig. 2. Effect of feeding strategy on dry cell weight and
specific growth rate during fed batch cultivation. Specific
growth rate (h-1) in un-induced () and induced processes () as
well as dry cell weight (g.l-1) in un-induced () and induced
processes ().

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Babaeipour et al.

fermentation condition resulted in an appropriate

expression level of GCSF.
Purity of final product was more than 99% (Fig. 6B),
which was validated by comparison with Neupogen
(Roche, Germany) and PD-Grastim (Pooyesh Darou,
Iran) as reference standards. In addition, two-step
washing procedure was sufficient to eliminate proteins
and DNA of host cell (data not shown). Obtaining
inspection chromatogram of TSK-GEL column for rhGCSF and Neupogen confirmed molecular weight of
rh-GCSF.

Fed-batch Stage

70
Induction

65
60
55
50
45
40

Batch Stage

35
30
0

Rh-GCSF concentration is also illustrated in Figure 4.

Acetate, glucose, ammonium, and phosphate
concentration were monitored during fed-batch
cultivation of recombinant E. coli (Fig. 5). The
concentration of all mentioned components was less
than the inhibitory level. It is obvious that specific
growth rate change before induction is negligible,
while it declines sharply after induction.
The expression of rh-GCSF before and after
induction in fed-batch culture of E. coli was
confirmed by SDS-PAGE and blotted GCSF
onto PVDF membrane (Fig. 6). Operating on
maximum achievable specific growth rate and suitable
Batch Stage

Fed-batch Stage
35
Induction
30

Plasmid Stability (%)

100

25

95

20
90
15
85

10

80

75

Rh-GCSF concentration (g l-1)

105

0
2

Induction

10 12 14 16 18

Time (h)
Fig. 3. Effect of feeding strategy on plasmid concentration on
the basis of CDW during fed-batch process.

Fed-batch Stage

14

10 12 14 16 18

Time (h)

Fig. 4. Effect of feeding strategy on plasmid stability and rhGCSF concentration during fed batch cultivation. Plasmid
stability (%) in un-induced () and induced processes () as
well as rh-GCSF concentration (g.l-1) in induced () and uninduced processes ().

12
Concentration (g l-1)

Plasmid concentration on the basis of

DCW (mg g-1)

Batch Stage

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10
8
6
4
2
0
0

10

12 14

16

18

Time (h)
Fig. 5. Effect of feeding strategy on concentration of the main
medium components (g l-1). Concentration of phosphate (),
glucose (), acetate (), and ammonium (), in induced
recombinant E. coli BL21 (DE3) [pET23a-hgcsf].

The Bradford's assay [24] and gel densitometry

analysis (Fig. 6B) showed that protein purification
yield was 400 mg protein per 1 g inclusion body
(40%). Based on the above results, it can be found out
that the purified protein (purity > 99%) in this article is
comparable with reference standards of Neupogen
(Roche, Germany) and PD-Grastim (Pooyesh Darou,
Iran). In comparison with other studies, the obtained
amount of recombinant protein in this article is one of
the highest productivity that has been recently reported
[28, 30-32]. Rh-GCSF expression and purification
yield were presented in the Table 1. The circular
dichroism spectra showed that the rh-GCSF was on par
with the reference standard of Neupogen (Fig. 7).
The circular dichroism spectrum of rh-GCSF in sodium
citrate buffer (pH 5) was consistent with the previously
reported structure (PDB ID code 1RHG), indicating an
alpha-helix content of 66-68% [3]. The circular
dichroism spectrum of Neupogen showed 67-70%
alpha-helix content. This result indicates that the
secondary structure of purified rh-GCSF is similar

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Efficient Process Development of rh-GCSF Production in E. coli

107

to Neupogen. The size exclusion chromatography

therefore, it is obvious that protein purification
successfully performed and purified protein has had no
dimer contamination. The increase in the growth of
HL-60 cells by rh-GCSF was similar to Neupogen.
Therefore, it can be concluded that the specific activity
of rh-GCSF is identical with the innovator product.
DISCUSSION

Fig. 6. SDS-PAGE analysis and Western blott of equivalent

gel of the total cell lysate of E. coli BL21 (DE3) [pET23ahgcsf]. (A) The end of the optimized fermentation. Lanes 1-6,
Coomassie-stained SDS-PAGE; Laned 7 and 8, Western blott of
equivalent gel using antibody to detect the rh-GCSF; lanes 1,
molecular weight markers (kDa); lane 2, total cell lysate at the
induction time (t = 0 h); lane 3, total cell lysate at t = 1 h; lane 4,
total cell lysate at t = 2 h; lane 5, total cell lysate at t = 3 h; lane
6, total cell lysate at t = 3.5 h. 1: The purity profile of rh-GCSF
expressed in E. coli. (B) SDS-PAGE (15%) analysis of purified
rh-GCSF showing a single protein band. Lane 1, Molecular
weight marker (#SM0431, Fermentas), lane 2, refolded rhGCSF; lane 3, purified rh-GCSF; lane 4, reference standard
(Neupogen); lane 5, reference Standard (PD-Grastim).

spectra of purified rh-GCSF and PD-Grastim (Fig.

8) showed that both proteins have a similar curve;

According to the data presented in Figure 2, specific

growth rates ranging from 0.55-0.4 h-1 were applied
before induction of rh-GCSF. Implementation of the
fed-batch process in higher specific growth rate before
induction led to higher biomass production and
consequently, higher productivity of GCSF, which was
in agreement with the data presented by other
researchers [14, 16, 28, 33].
Figure 2 illustrates that during feeding, the specific
growth rate was decreased, which may be because of
(1) inhibitory effect of acetate accumulation, (2)
oxygen transfer limitation due to high cell density and
accumulation of antifoam at the end of the
fermentation process [12, 34], (3) stress response
related to induction of recosmbinant protein
overexpression in E. coli and environmental stresses
such as very high mixing rate [8]. This Figure shows a
sharp decrease in specific growth rate after induction
because of the increasing metabolic burden of cells by
recombinant gene overexpression [12, 34]
The results of plasmid content (Fig. 3) showed that in
the employed fed-batch technique, high cell density has
no negative effect on plasmid content. Furthermore,
Plasmid stability is one of the most important issues
affecting the productivity of recombinant protein
production in E. coli fed-batch cultivation [31, 32].
Although it has been difficult to find real trends in
relationship between plasmid stability and specific
growth rate in most cases, change in this parameter
seems to be a function of the specific growth rate in the
fed-batch. It has found that plasmid stability is reduced
by decreasing growth rate, primarily because growth
rate of plasmid-containing cells is less than the
plasmid-free cells. [13, 31, 32]. Therefore, it can be
expected that plasmid stability increases by increasing
the specific growth rate (Fig. 4).

Table 1. Protein expression and purification yield

Dry cell weight

Rh-GCSF concentration

Rh-GCSF yield

None-induced fed-batch

142 (g/lit)

---

---

Induced fed-batch
Purification

127 (g/lit)
---

32 (g/l)
400 (mg/g IB)

1.83 (mg.l-1.h-1)
40%

IB, inclusion body

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Babaeipour et al.

Fig. 7. Circular dichroism spectra of rh-GCSF, and

Neupogen. Circular dichroism spectra of rh-GCSF (black) and
Neupogen (gray) are shown. The mean residue ellipticity of the
proteins (10 mM) were measured at 22C in 10 mM citrate
buffer at pH 5.

At high cell density, acetate formation and

accumulation are main challenges during recombinant

Iran. Biomed. J., April 2015

protein production [10, 28] that can be minimized by

controlling the specific growth rate below a certain
value (depending on strain) [12]. In this study, by
exponential feeding strategy, specific growth rate was
controlled below critical value; therefore, acetate
concentration was controlled under inhibitory level
(less than 5 g l-1 for acetate) [35] (Fig. 5). By this
feeding strategy, glucose concentration was also
maintained simultaneously at a suitable concentration
range without any starvation and accumulation of
glucose (Fig. 5). Dissolved oxygen concentration was
maintained higher than least quantity that was reported
throughout fed-batch mode (more than 6%, v/v air
saturation) [36].
In this study, human recombinant protein expression
was carried out in developed fed-batch culture of E.
coli (data not shown). Therefore, in comparison with
other studies, the cultivation time was decreased, while
cell density and rh-GCSF concentration were increased
significantly [6, 13, 25, 30, 31]. Overall, the productivity of GCSF was higher than that reported by other
researchers [34].

Fig. 8. Size exclusion chromatography (SEC) spectra of rh-GCSF, and Neupogen. SEC spectra of rh-GCSF (A) and Neupogen (B)
are shown. The SEC has done with TSK-GEL G3000SWXL (300 mm 7.8 mm) column. Moving phase contain 1mM K2HPO4Na2HPO4 in water at pH 6.2.
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Efficient Process Development of rh-GCSF Production in E. coli

Figure 6A shows the SDS-PAGE of total cell

proteins and Western blot of equivalent gel after
induction at a cell density of 75 g l-1 DCW with 2 mM
IPTG as inducer. Fraction of rh-GCSF remarkably
increases by passing time after induction, and
percentage of expressed recombinant protein to total
protein at the end of the process is 45.
This process has the following advantages resulting
in the higher percentage of recombinant protein
expression to total protein: (1) reduction of process
time, (2) decrease of by-products accumulation,
especially acetate, (3) increase of plasmid stability, (4)
suitable concentration range of nutrients, such as
glucose, ammonium and phosphate during fed-batch
cultivation [7, 13, 14, 26, 37].
Applying modified exponential feeding strategy for
fed-batch culture of recombinant E. coli resulted in an
increase in attainable specific growth rate, reduce of
process time, and maximum productivity. Therefore,
this strategy can successfully be applied to enhance the
production of any recombinant proteins in E. coli or
other expression systems.
Based on the above results, it can be found that rhGCSF protein isolated in this study is highly pure and
comparable with the innovator products, Neupogen
and PD-Grastim. These properties serve as a basis for
comparison of process reproducibility, creating the
range of conditions to stabilize the protein during
production and storage as well as for identifying
characteristics valuable for monitoring stability during
long-term storage.
In the present study, we developed an efficient
procedure for production and purification of rh-GCSF
in E. coli by using a new developed method. According
to the available data, result of this article is one of the
highest YP/X and productivity that has been reported for
any human recombinant protein expressed in E. coli
[27, 28, 30, 34, 38].

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REFERENCES
1.

2.

3.

Tian H, Liu G, Gao XD, Yao WB. Optimization of autoinduction medium for G-CSF production by Escherichia
coli using artificial neural network coupled with genetic
engineering. World J Microbiol Biotechnol. 2012 Nov;
29(3):505-13.
Sharifi Tabar M, Habashi AK, Rajab Memari H. Human
granulocyte-colony stimulating factor (rh-GCSF)
expression in plastids Lactuca saive. IBJ. 2013 July;
17(3):158-64.
Ko JH, Kim KH, Pang MG, Koo JS, Fang N, et al.
Production of biologically active human granulocyte
colony stimulating factor in the milk of transgenic goat
production of biologically active human granulocyte
colony stimulating factor in the milk of transgenic goat.
Transgenic Res. 2000 May; 9(3):215-22.

16.

17.
18.

19.
20.

109

Orawan K, Sunutcha S. Strategies for production of

active eukaryotic proteins in bacterial expression
system. Asian Pac J Trop Biomed; 2012 Feb; 2(2):15962.
Zhao Y, He W, Liu WF, Liu CC, Feng LK, Sun L, et al.
Two distinct states of Escherichia coli cells that
overexpress the recombinant heterogeneous galactosidase.
J
Biol
Chem.
2012
Feb.
http://www.jbc.org/cgi/doi/10.1074/jbc.M111.327668.
Choi JH, Keum KC, Lee SY. Production of recombinant
proteins by high cell density culture of Escherichia coli.
Chem Eng Sci. 2006 Feb; 61(3):876-85.
Jana S, Deb JK. Strategies for efficient production of
heterologous proteins in Escherichia coli. Appl
Microbiol Biotechnol. 2005 May; 67(3):289-98.
Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of
glucose supply strategy on acetate accumulation,
growth, and recombinant E. coli BL21 (lDE3) and E.
coli JM109. Biotechnol Bioeng. 1996 Feb;
20;49(4):421-8.
Heyland J, Blank LM, Schmid A. Quantification of
metabolic limitations during recombinant protein
production in Escherichia coli. J Biotechnol. 2011 Sep;
155(2):178-84.
Babaeipour V, Shojaosadati SA, Khalilzadeh R,
Maghsoudi N, Tabandeh F. A proposed feeding strategy
for overproduction of recombinant proteins by E. coli.
Biotechnol Appl Biochem. 2008 Feb; 49(Pt 2):141-7.
Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer
WD. Simple fedbatch technique for high cell density
cultivation of Escherihcia coli. J. Biotechnol. 1995 Feb;
39(1):59-65.
Lee SY. High cell-density culture of Escherichia coli.
Trends Biotechnol 1996 Mar; 14(3):98-105.
Gregory ME, Turner C. Open-loop control of specific
growth rate in fed-batch cultures of recombinant E. coli.
Biotechnol Tech. 1993 Des; 7(12):889-94.
Sanden AM, Prytz I, Tubulekas I, Forberg C, Le H,
Hektor A, et al. Limiting factors in Escherichia coli fedbatch production of recombinant proteins. Biotechnol
Bioeng. 2003 Jan; 81(2):158-166.
Hoffmann F, Heuvel JVD, Zidek N, Rinas U.
Minimizing inclusion body formation during
recombinant protein production in Escherichia coli
atbench and pilot plant scale. Enzyme Microb Technol.
2004 Mar; 34(3-4):235-41.
Oh JS, Kim BG, Park TH. Importance of specific
growth rate for subtilisin expression in fed-batch
cultivation of Bacillus subtilis spoIIG mutant. Enzyme
Microb Technol. 2002 May; 30(6):747-51.
Vallejo LF, Ursula R. Strategies for the recovery of
active proteins through refolding of bacterial inclusion
body proteins. Microb Cell Fact. 2004; 3:11.
Batas B, Schiraldi C, Chaudhuri JB. Inclusion body
purification and protein refolding using microfiltration
and size exclusion chromatography. J Biotechnol. 1999
Feb; 68(2-3):149-58.
Basu S, Dunn A, Ward A. G-CSF: Function and modes
of action (Review). Int J Mol Med. 2002 Jul; 10(1):310.
Enfors SO, Jahic M, Rozkov A, Xu B, Hecker M,
Jrgen B, et al. Physiological responses to mixing in

http://IBJ.pasteur.ac.ir

110

21.

22.

23.

24.

25.
26.

27.

28.

29.

Babaeipour et al.

large scale bioreactors. J. Biotechnol. 2001 Feb;

13;85:175-85.
Fallah MJ, Akbari B, Saeedinia AR, Karimi M, Vaez M,
Zeinoddini M, et al. Overexpression of Recombinant
Human Granulocyte Colony-Stimulating Factor in E.
coli. IJMS. 2003 Sep; 28(3):131-4.
Panda AK, Khan RH, Rao KBCA, Totey SM. Kinetic of
inclusion body production in batch and high cell density
fed-batch culture of Escherichia coli expressing ovine
growth hormone. J. Biotechnol. 1999 Oct; 8;75(23):16172.
Burnette WN. Western Blotting: Electrophoretic
transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and
radiographic detection with antibody and radioiodinated
protein A. Anal Biochem. 1981 Apr; 112(2):195-203.
Bradford MM. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle protein dye-binding. Anal Biochem. 1976
May;7;72:248-54.
Gershoni JM, Palade GE. Protein blotting: Principles
and applications. Anal Biochem. 1983 May; 131(1):115.
Curless CE, Pope J, Tsai L. Effect of pre-induction
specific growth rate on recombinant alpha consensus
interferon synthesis in Escherichia coli. Biotechnol
Prog. 1990 Mar-Apr; 6(2):149-52.
Rao Dasari VK, Are D, Joginapally VR, Mangamoori
LN, Adibhatla KSBR. Optimization of the downstream
process for high recovery of rhG-CSF from inclusion
bodies expressed in Escherichia coli. Process
Biochem.2008 May; 43(5):566-75.
Codevilla CF, Brum L, Oliveira PR, Dolman C, Rafferty
B, Dalmora LS. Validation of an SEC-HPLC method for
the analysis of rhG-CSF in pharmaceutical formulations.
J Liq. Chromatogr RT. 2004; 27(17):2689-98.
Khalilzadeh R, Shojaosadati SA, Bahrami A,
Maghsoudi N. Over-expression of recombinant human
interferon-gamma in high cell density fermentation of

30.

31.

32.

33.

34.

35.

36.
37.

38.

Iran. Biomed. J., April 2015

Escherichia coli. Biotechnol Lett. 2003 Dec; 25:198992.

Yang SY, Bae CS, Lee J. Production of recombinant
human granulocyte-colony-stimulating factor in high
cell density yeast cultures. Biotechnol Lett. 1997 Jul;
19(7):655-9.
Rao DVK, Narasu ML, Rao AKSB. A purification
method for improving the process yield and quality of
recombinant human granulocyte colony-stimulating
factor expressed in Escherichia coli and its
characterization. Biotechnol Appl Biochem. 2008;
50:77-87.
Vanz ALS, Renard G, Palma MS, Chies JM, Dalmora
SL, Basso LA, et al. Human granulocyte colony
stimulating factor (hG-CSF): cloning, overexpression,
purification and characterization. Microb Cell Fact.
2008 Jan; 7(13):.
Koom TY, Park TH. Increased production of
recombinant protein by Escherichia coli deficient in
acetic acid formation. J Microbiol Biotechnol. 1999;
9:789-93.
Filomena S, Joao AQ, Fernanda C. Domingues,
Evaluating metabolic stress and plasmid stability in
plasmid DNA production by Escherichia coli. Biotech
Adv. 2012 Jan; 30:691-708.
Kim BS, Lee SC, Lee SY, Chang YK, Chang HN. High
cell density fed-batch cultivation of Escherichia coli
using exponential feeding combined with pH-stat.
Bioprocess Biosyst Eng. 2004 Feb; 26(3):147-50.
Kleman GL, Stroh WR. Developments in high cell
density and high productivity microbial fermentation.
Curr Opin Biotechnol. 1994 Apr; 5(2):180-6.
Yim SC, Jeong KJ, Chang HN, Lee SY. High-level
secretory production of human G-CSF by fed-batch
culture of recombinant Escherichia coli. Bioproc
Biosyst Eng. 2001 Nov; 24:249-54.
Lee J, Lee SY, Park S. Fed-batch culture of Escherichia
coli W by exponential feeding of sucrose as a carbon
source. Biotechnol Tech. 1997 Jan; 11(1):59-62.

http://IBJ.pasteur.ac.ir