Biotechnology Letters 25: 1989–1992, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
1989
Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli R. Khalilzadeh, S.A. Shojaosadati∗ , A. Bahrami & N. Maghsoudi Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modarres University, Tehran, P.O. Box 14155-4838, Iran ∗ Author for correspondence (Fax: +9821/8005040; E-mail:
[email protected]) Received 13 August 2003; Revisions requested 27 August 2003; Revisions received 30 September 2003; Accepted 2 October 2003
Key words: Escherichia coli, fed-batch fermentation, plasmid stability, recombinant human interferon-gamma
Abstract Human interferon-gamma (hIFN-γ ) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fedbatch process. Cell density of recombinant E. coli was reached to 100 g dry wt l−1 under both constant (0.12 h−1 ) and variable (0.12–0.52 h−1 ) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-γ were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-γ were 0.35 ± 0.02 g rhIFN-γ g−1 dry cell wt and 0.9 ± 0.05 g rhIFN-γ l−1 h−1 in the variable specific growth rate fed-batch process, respectively.
Introduction Human interferon-gamma (hIFN-γ ) is a glycosylated protein with a total molecular size of 25 kDa and composed of 143 amino acid residues (Farrar & Schreiber 1993). Recombinant hIFN-γ (rhIFN-γ ) expressed in Escherichia coli is not glycosylated with a molecular size of 17 kDa but is still physiologically active (Zhang et al. 1992). High cell density cultivation (HCDC) techniques have developed for the enhancement of productivity of recombinant products (Riesenberg & Guthke 1999, Lee 1996). It was commonly observed that the specific yield of recombinant products obtained by HCDC considerably decreased with the increasing cell density in comparison to that obtained in simple batch fermentation (Panda et al. 1999, Yang et al.1992). Plasmid instability is one of the reasons for the decreasing of the specific yield (Yang et al. 1992, Yoon & Kang 1994, Khalilzadeh et al. 2002). Genetic and environmental factors may influence plasmid stability (Zhang et al. 1996). In this research, the effect of specific growth rate on plasmid stability and specific yield of rhIFN-γ
during fed-batch fermentation of recombinant E. coli BL21(DE3) was studied. We have also developed a simple fed-batch strategy to increase the specific yield of rhIFN-γ expression using HCDC techniques.
Materials and methods Microorganism and vector Escherichia coli strain BL21(DE3) (Novagen, Inc.) was used as the host for rhIFN-γ expression. This strain was transformed with pET3a inducible expression vector (Novagen, Inc), in which hIFN-γ gene (Noor Research & Educational Institute, Tehran, Iran) was inserted into the NotI and NdeI sites (Khalilzadeh et al. 2002). Media and cultivation condition Recombinant E. coli was cultivated in LB (Luria– Bertani) agar and defined media (M9 modified medium) consisting 10 g glucose, 12.8 g Na2 HPO4 · 7H2 O, 3 g KH2 PO4 , 0.5 g NaCl, 1 g NH4 Cl,
1990 0.5 g MgSO4 · 7H2 O, and 1 ml trace element solution per liter. The trace element solution consisted 2.8 g FeSO4 · 7H2 O, 2 g MnCl2 · 4H2 O, 2.8 g CoSO4 · 7H2 O, 1.5 g CaCl2 · 2H2 O, 0.2 g CuCl2 · 2H2 O, and 0.3 g ZnSO4 · 7H2 O per liter of 1 M HCl. The glucose and MgSO4 solutions were sterilized separately to make the feeding solution with 750 g glucose l−1 , 20 g MgSO4 · 7H2 O l−1 and 5 ml trace element solution l−1 . The fed-batch fermentation was carried out in a 2 l bench-top bioreactor (INFORS AG, Switzerland) with the working volume of 1 l. The initial batch culture was started by 100 ml of overnight-incubated seed culture (0.4–0.6 dry cell wt l−1 ). The pH was controlled at 7 ± 0.05 by the addition of 25% (w/w) NH4 OH or 1M H3 PO4 . Ammonia was maintained in range of 0.1 and 1.5 g l−1 . Dissolved O2 was controlled at 1.5– 2.2 mg l−1 (20–30% saturation) by control of both airflow and stirrer speed. During the fed-batch phase, the inlet air was enriched with pure O2 and foam was controlled by the addition of silicon-antifoaming reagent. After depletion of the initial glucose in the batch medium, feeding was initiated and the flow rate was increased stepwise based on exponential feeding strategy (Ejiofor et al. 1996). Analytical procedures The optical density (OD) of the culture was measured at 600 nm and converted to the dry cell weight (cells dried at 105 ◦ C to a constant weight) by an appropriate calibration curve. Glucose and ammonia were analyzed enzymatically with glucose and ammonia kits (ChemEnzyme Co.). Acetate was assayed using an enzymatic analysis kit (Boehringer Mannheim/R-Biopharm). Expression of rhIFN-γ was determined by SDS-PAGE on 12.5% (w/v) polyacrylamide gels stained with 0.1% (w/v) Coomassie Brilliant Blue R250 and quantified by densitometry. Total soluble protein was analyzed by Bradford method and rhIFN-γ analyzed by ELISA assay (Maghsoudi et al. 2001). The plasmid stability was tested by aseptically sampling from the bioreactor at different dry cell weight, preparation of serial dilution by sterile solution of 9 g NaCl l−1 and plating on LB agar plates without ampicillin. All of the colonies were transferred on LB plates supplemented with 100 mg ampicillin l−1 by replica plating method.
Results and discussion Constant specific growth rate fed-batch process By using experimental data from various fed-batch cultures of E. coli BL21(DE3) harboring pET3ahIFN-γ vector, specific growth rate of 0.12 h−1 was selected as the set point of the specific growth rate in the exponential feeding strategy, to avoid formation of growth inhibitory metabolites particularly acetate. The final cell density reached greater than 100 g dry cell wt l−1 after 36 h cultivation (Figure 1). Although the used expression vector system is inducible, it was shown relatively high background expression and plasmid instability in this process (Figure 1). Since expression of heterologous protein is burden on the host cell, it causes significant decreasing in growth rate and plasmid stability. Variable specific growth rate fed-batch process By using experimental data from various fed-batch cultures of recombinant E. coli BL21(DE3) in the glucose unlimited condition was correlated the appropriate equation for decreasing of specific growth rate (Equation (1)). µ = −0.004(t − t0 )2 − 0.03(t − t0 ) + 0.52 0.12 < µ < 0.52,
(1)
where µ (h−1 ) is the specific growth rate, t (h) is the time of fermentation and t0 (h) is the time of start feeding. By using this equation for determination of feeding rate, fermentation time was decreased and plasmid stability increased compare with the constant specific growth rate fed-batch process. In this feeding strategy, the formation of growth inhibitory by-products especially acetate was also below inhibitory concentrations (Figure 2). Variation of plasmid stability with change in specific growth rate, medium composition, dissolved O2 , and temperature have been observed (Panda et al. 1999, Zhang et al. 1996, Yoon & Kang 1994, Yang et al. 1992); however, these results indicate that using of variable specific growth rate is more convenient than the constant specific growth rate in fed-batch process for high cell density cultivation of recombinant E. coli BL21(DE3). Production of rhIFN-γ in HCDC Recombinant E. coli BL21(DE3) was cultivated in a 2-l bench top bioreactor with a working volume of 1 l
1991
Fig. 1. Growth kinetics of E. coli BL21(DE3) harboring pET3a-hIFN-γ at constant specific growth rate (0.12 h−1 ) fed-batch process in a 2-l bench top bioreactor containing 1 l of defined M9 modified medium.
Fig. 2. Growth kinetics of E. coli BL21(DE3) harboring pET3a-hIFN-γ at variable specific growth rate (0.12–0.52 h−1 ) fed-batch process in a 2-l bench top bioreactor containing 1 l of defined M9 modified medium.
using fed-batch process with variable specific growth rate feeding strategy. When the cell density of the recombinant E. coli BL21(DE3) was reached about 50 g dry cell wt l−1 , adding of IPTG to give 3 mM into the fermenter induced efficiently expression of rhIFN-γ (Figure 3). This overproduction of heterologous protein (Figure 3) leads to decreases in growth rate and inhibition of cell mass production, as reported by other researchers (Panda et al. 1999, Yoon & Kang 1994).
The maximum amount of rhIFN-γ was achieved after 5 h of post induction. The final cell density was reached about 58 ± 2 g dry wt l−1 at 23 ± 1 h. Also the final specific yield and overall productivity of rhIFNγ were obtained as 0.35 ± 0.02 g rhIFN-γ g−1 dry cell wt and 0.9 ± 0.05 g rhIFN-γ l−1 h−1 , respectively. Which are the highest specific yield and overall productivity reported for recombinant protein in the HCDC techniques.
1992 Acknowledgements The authors acknowledge the support of some part of this research by Noor Research & Educational Institute and Shafa-e-Sari Antibiotic Producing Company, Iran.
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Fig. 3. Growth kinetics of E. pET3a-hIFN-γ at variable specific fed-batch process with induction at IPTG, in a 2-l bench top bioreactor modified medium.
coli BL21(DE3) harboring growth rate (0.12–0.52 h−1 ) 50 g DCW l−1 by 3 mM of containing 1 l of defined M9
