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A simple fed-batch process was developed for high cell density cultivation of Escherichia coli BL21 (DE3) [pET23a-hgcsf] Expressing human granulocyte-colony stimulating factor (GCSF). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h-1 at the beginning of feeding and then it was reduced to 0.4 h-1 at induction time. The final concentration of biomass and GCSF was reached to 126 g(DCW).l-1 and 32.1 g.l-1 after 17.5 h which is the highest dry cell concentration with recombinant protein expression in E. coli up to now; also the yield YP/X and overall productivity of rh-GCSF were obtained 254 mg (rh-GCSF).g-1 DCW and 1.83 g (rh-GCSF) l-1 h-1, respectively. According to available data this is one of the highest YP/X and productivity that has been reported for any human recombinant protein which is expressed in E. coli.
Keywords: rh-GCSF, E. coli, Fed Batch Cultivation, Yield, Productivity, High Cell Density
Human granulocyte colony-stimulating factor (h-GCSF) is a single chain polypeptide with a total molecular size of 18.8 kDa and is composed of 174 amino acid residues. GCSF is hemopoietic growth factor which plays an important role in stimulating proliferation, differentiation, and functional activation of blood cells. It contains a free cysteine at position 17 and two intramolecular disulfide bonds which are required for GCSF bioactivity (Basu et al. 2002, Hill et al. 1993).
E. coli is one of the most widely used hosts for the production of heterologous proteins (Choi et al. 2006, Jana and Deb 2005, Shiloach et al. 1996), because of its simple nutrient requirement, high growth rate and its well known molecular genetics and physiology.
Because most proteins are accumulated intracellularly in recombinant E. coli, productivity is proportional to the final cell density and the specific productivity (i.e. the amount of product formed per unit time). For this reason, the high cell density cultivation (HCDC), which is attainable in fed-batch processes, becomes important for cultivation of E. coli (Lee 1996). In the fed-batch system, feeding strategy is critical to the success of HCDC, because it affects the metabolic pathway fluxes, and consequently affects the maximum attainable cell concentration, the specific productivity of recombinant proteins, and formation of by-products (Babaeipour et al. 2008, Choi et al. 2006, Korz et al. 1995, Lee 1996, Yim et al. 2001).
Various nutrient feeding strategies have been employed for the HCDC of E. coli (Lee 1996). Exponential feeding method is probably one of the most widely used methods for growing cells to high cell densities in the fed-batch cultivation; since, in this methodology it is easy to implement the process and manipulate of specific growth rate (Cheng et al. 2002, Choi et al. 2006, Gregory and Turner 1993, Lee 1996).
It has been shown that the specific productivity of recombinant proteins obtained by HCDC is generally lower than that obtained by batch culture (Choi et al. 2006). A possible reason is that many fed-batch processes are run at low specific growth rates and under substrate limited conditions before and after induction; and, consequently most available carbon and energy sources have to be used for maintenance requirements (Sanden et al. 2003). On the other hand, the higher specific growth rate during production (Shin et al. 1997) or even before induction (Curless et al. 1994, Hoffmann et al. 2004) can increase recombinant protein production, presumably via higher ribosome content (Hoffmann et al. 2004). Also achieving substrate non-limited conditions can reduce stress responses before and after induction (Hoffmann et al. 2004, Sanden et al. 2003, Vidal et al. 2005). Therefore, manipulating specific growth rate, as a key process parameter that represents the dynamic behavior of microorganisms during fermentation, at an appropriate range in a fed-batch process can provide a desirable metabolic condition and results in maximum productivity (Koo and Park 1999, Oh et al. 2002, Panda et al. 1999, Sanden et al. 2003).
Therefore, in this study an attempt was made to obtain higher yield and productivity by keeping specific growth rate at a maximum attainable level during the exponential feeding of fed-batch cultivation. Also, the effect of feeding strategy on acetate production as the main byproduct, phosphate, ammonium, and glucose concentration in the cultivation medium, plasmid stability, total process time, and glucose and ammonium consumed in the fed-batch process were studied during HCDC of recombinant E. coli BL21 (DE3) [pET23a-hgcsf].
Materials and methods
Microorganism and vector system
Escherichia coli strain BL21 (DE3) (Novagen, Inc.) was used as the host for rh-GCSF expression. This strain was transformed with a commonly available plasmid, pET23a inducible expression vector (Novagen, Inc.), in which the rh-GCSF gene (Fallah et al. 2003) was inserted into the NotI and NdeI sites. Host cells were transformed with the plasmid using the calcium chloride procedure. Transformed cells were spread on several LB agar plates containing 100 mg/l ampicilin.
Media and solutions
LB (Luria-Bertani) medium was used for plate cultivation of E. coli strain BL21 (DE3) [pET23a-hgcsf] and M9 medium was used for preparation of seed culture. M9 modified medium consisted of 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 contained 2.8 g FeSO4.7H2O, 2g MnCl2.4H2O ØŒ2.8 g CoSO4.7H2O, 1.5 g CaCl2.2H2O, 0.2 g CuCl2.2H2O, and 0.3 g ZnSO4.7H2O g per liter in 1M HCl. Fed-batch cultivation were 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 50-1200 rpm.
Analytical procedure consists of three main stages; cell cultivation and ingredient measurements, cell harvesting, and recombinant protein assessment and measurement by electrophoresis and Western blotting.
Cell growth was monitored by measuring culture turbidity and dry cell weight (DCW). Turbidity was determined by measuring optical density (OD) at 600m in each 10 min. Samples were diluted with NaCl solution (9 g/l) to obtain an OD600 between 0.2 and 0.5. In order to determine dry cell weight (DCW), 5 ml of broth was centrifuged at 9000 rpm for 10 min, washed twice with deionized water, and dried at 105 °C to constant weight. Glucose, ammonia, phosphate and acetate were analyzed enzymatically by using the appropriate kits (ChemEnzyme CO., I.R. Iran; Boehringer Mannheim/R-Biopharm, Germany).
Samples from cultivation were directly chilled on ice with further centrifugation (3min, 13000 rpm, 4 °C) and the supernatant was collected by direct filtration through a 0.2 µm disk filter. Afterwards, the samples were stored at -20°C for further analysis.
The expression level of rh-GCSF was determined by SDS-PAGE using poly-acrylamid 12.5 % (w/v) (Weber et al. 1972). Gels were stained with Coomassie brilliant blue R250, and then quantified by gel densitometer. Total soluble protein was analyzed by Bradford method (Bradford 1976). Separated proteins on the SDS-PAGE gels were transferred and blotted on the PVDF membrane for recognizing the exact existence of rh-GCSF (Burnette 1981, Gershoni and Palade 1983). Expressed recombinant protein was determined and quantified by SDS-PAGE, Western blotting, and Bradford methods. At the end of cultivations, some samples were collected for further purification and analysis by recently developed method which were used in our previous study (Babaeipour et al. 2009).
The stability of the plasmid in the recombinant E. coli strain was determined by sampling aseptically from the bioreactor at different cell densities. When required, fermentation broth samples, diluted with 9% (w/v) NaCl were plated on to LB agar plates with and without ampicillin (three replicates for each case). The fraction of plasmid-containing cells was calculated as the average ratio of viable colonies on LB with ampicillin to those on LB without the antibiotic (Panda et al. 1999). Plasmids were exctracted from cells by plasmid DNA extraction kit (Roche, Germany) and were quantified by measuring absorption at 260 nm.
A batch culture was initially established by the addition of 100 ml of an overnight-incubated seed culture (CDW = 0.4 - 0.6 g l-1) to the bioreactor containing 900 ml of M9 modified medium. The pH was maintained at 7 by the addition of 25% (w/v) NH4OH or 3 M H3PO4 solutions. Dissolved oxygen was controlled at 30-40% (v/v) of air saturation by controlling of both the inlet air (which was enriched with pure oxygen) and agitation rate. Foam was controlled by the addition of silicon-antifoaming 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 stepwise based on the exponential feeding strategy with maximum attainable specific growth rate during fed-batch cultivation. The exponential feeding was determined by the following equation (Eq. (1)) (Korz et al. 1995):
M (t) = F (t) S0 = [ m + µ(t)/Yx/s ] S0 V0 X0 exp [ ∫t0t µ(t) dt] (1)
where V0 is the volume of the medium in the bioreactor (l), X0 is the biomass concentration at the start of feeding g(DCW) l-1, t is the time (h), μ 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 the feeding rate (l h-1), M(t) is the 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 is corrected by the turbidity of the taken samples in each 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. In order to develop 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. Maximum oxygen transfer is obtained by increasing the impeller rotation speed from 400rpm to 1200rpm during the process. 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 the 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; also, temperature of the process was maintained at 37 °C. The level of phosphate added at the beginning of fed-batch was sufficient until end of process. Acetate and glucose concentrations were controlled manually at intervals 10 min.
Based on the previous results regarding variable specific growth rate feeding strategies (Khalilzadeh et al. 2003), experiments were designed to obtain a feeding strategy which would lead to the highest attainable specific growth rate before induction. In order to perform such experiments, feeding rate was increased by maintaining the glucose concentration within a permissible range and by using the maximum oxygen transfer capacity of the bioreactor.
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 non-limited conditions (Eq. (2)):
µ(t)= -0.967 (t-t0)3 + 36.21(t-t0)2-432.2 (t-t0)+167.2,
R2 = 0.97, 0.04 < µ < 0.54 (2)
t0 = time at the start of feeding
Eqs. (1) and (2) were used to determine the feeding rate in the fed-batch process. Fig. 1 shows the results of using this feeding strategy on fed-batch cultures of un-induced recombinant E. coli BL21 (DE3) [pET23a-hgcsf].
Cell dry weight and specific growth rate in un-induced and induced fed-batch are illustrated in Fig. 1. In un-induced process cell growth terminated after 17.5 ± 0.5 h, when a cell density 145 ± 5 g (DCW) l-1 was reached. According to the results, cell density reached to the 75 g (DCW) l-1 during the first 14 h of the fed-batch operation, where the variation in the specific growth rate was within in a rang of 0.55-0.43 h-1; And, specific growth rate decreases from 0.55 to 0.04 h-1 during feeding. Also, Fig. 1 shows more decrease of specific growth rate in the induced fed-batch
As it is illustrated in Table 1, By applying this feeding strategy in induced process, maximum cell density and GCSF concentration after 17.5 ± 0.5 h were 125 ± 5 g(DCW) l-1 and 32 ± 1 g(GCSF) l-1, respectively. Also the maximum YP/X and productivity of GCSF were 250 ± 10 mg(GCSF) g-1 (DCW) and 1.83 ± 0.05 g(GCSF) l-1 h-1, respectively.
As it is demonstrated in Fig 2, plasmid concentration remains constant before the induction and increases after induction. And, Fig. 3 shows that plasmid stability was maintained at the highest level until the end of the fed-batch process. A slight decrease in plasmid stability after induction is due to an increase in the metabolic burden of cells arising from recombinant gene expression. Also, rh-GCSF concentration is illustrated in the same figure (Fig. 3).
Figs. 4 show concentrations of acetate, glucose, ammonium, and phosphate during fed-batch cultivation in induced process. All of components are lower than the inhibitory concentrations of these chemicals pre- and post-induction.
Fig. 5 shows yield for biomass and protein production pre- and post-induction. It is clear that variation in yield before induction is negligible for biomass, but yield of biomass production declines sharply after induction; also, low increase in protein yield can be seen before the induction, but there is a sudden and sharp increase after induction.
Fig. 6 shows a typical SDS-PAGE of total cell proteins pre- and post-induction in fed-batch culture; and, blotted GCSF onto PVDF membrane. The results of SDS-PAGE profile and blotted membrane reveal a considerable expression of GCSF in fed batch process which operates on maximum attainable specific growth rate.
According to data presented in Fig. 1, specific growth rates ranging from 0.55 to 0.4 h-1 were applied before induction. The selection of the higher specific growth rate before induction resulted in higher biomass production and consequently led to a high specific yield of GCSF, which was in agreement with the data presented by other researchers (Curless et al. 1994, Hellmuth et al. 1994, Hoffmann et al. 2004, Khalilzadeh et al. 2003, Oh et al. 2002, Sanden et al. 2003, Vidal et al. 2005).
Fig. 1 indicates that during feeding, specific growth rate decreases which may be due to (1) accumulation of toxic by-products such as acetate, and (2) limitation of oxygen transfer due to increase in cell density and presence of more antifoam at the end of the process (Koo and Park 1999, Lee 1996, Shiloach and Fass 2005), (3) Induction stress response of recombinant E. coli environmental stresses such as pure oxygen and very high mixing rate (Castan et al. 2002, Enfors et al. 2001). Fig. 1 shows more decrease of specific growth rate in the induced fed-batch which is due to the increasing metabolic burden of cells by recombinant gene expression (Lee 1996, Neubauer et al. 2003, Shiloach and Fass 2005).
The results of plasmid content (Fig. 2) show that in the employed fed-batch technique, high cell density has no negative effect on plasmid content. Also, Plasmid stability is one of the most important issues affecting the productivity of recombinant protein production in E. coli fed-batch cultivation (Neubauer et al. 2003). In fed-batch cultivation the change in this parameter seems to be a function of the specific growth rate, although it has been difficult to find real trends in most cases. It has generally been observed that plasmid stability reduces with decreasing growth rate, primarily because the relative growth rate advantage of plasmid-free cells over those containing plasmids decreases under these conditions (Gregory and Turner 1993, Neubauer et al. 2003). Hence, it can be expected that by increasing the specific growth rate, plasmid stability increases (Fig 3).
At high cell density, acetate formation and accumulation is a major challenge during recombinant protein production (Kleman and Strohl 1994) which can be minimized by controlling the specific growth rate below a certain value (depending on strain and medium composition) (Lee 1996). By controlling and keeping specific growth rate below the critical value in all of experiments, acetate concentration were lower than the reported inhibitory growth limit (less than 5 g l-1 for acetate) (Figs. 4). Also, glucose concentration was maintained at a permissible range simultaneously, without any starvation and accumulation of glucose (Figs. 4). The dissolved oxygen concentration was kept higher than minimum amount that was reported during fed-batch (more than 6%, v/v air saturation) (Lee et al. 1997).
Summarization of rh-GCSF production in developed fed batch process is depicted in Table 2; also, the fraction of GCSF increase with time after induction is shown in Table 1. Although, human recombinant protein expression is carried out in the developed fed-batch process (Fig 5), in comparison with data reported by other researchers, the cultivation time in this process decreases while cell density, GCSF concentration, and biomass increase significantly (Choi et al. 2006, Kleman and Strohl 1994, Lee et al. 1997, Lee 1996, Panda et al. 1999, Shiloach et al. 1996). Overall productivity of GCSF was higher than those reported by other researchers (Yim et al. 2001).
Fig. 6 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 with time after induction; and, percentage of expressed recombinant protein due to total protein in the in the end of the process is 45%.
This process has various advantages which resulted in the higher percentage of recombinant protein expression due to total protein, and these advantages can be listed as (1) reduce of process time, (2) decrease in accumulation of by-products, especially acetate, (3) increase in plasmid stability, (4) presence of nutrients (glucose, ammonium and phosphate) at a suitable concentration range during fed-batch cultivation, and (5) provision of suitable physiological and metabolic conditions during fed-batch cultivation (Curless et al. 1994, Gregory and Turner 1993, Hellmuth et al. 1994, Jana and Deb 2005, Kleman and Strohl 1994, Koo and Park 1999, Lee et al. 1997, Neubauer et al. 2003, Oh et al. 2002, Sanden et al. 2003, Shiloach and Fass 2005, Shiloach et al. 1996).
Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli was established to achieve maximum attainable specific growth rate pre- and post induction and to minimize fed-batch duration and maximize product yield and productivity; The results of this method shows that by using a proper feeding strategy the productivity of biomass and recombinant protein will increase in comparison with earlier reported results (Babaeipour et al. 2008, Khalilzadeh et al. 2003, Yim et al. 2001). Therefore this strategy can successfully be applied to enhancing the production of any recombinant proteins in E. coli, or other expression systems.