Screening for the optimal induction parameters for periplasmic producing interferon-α2b in Escherichia coli (original) (raw)
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Biotechnology and Applied Biochemistry, 2010
Induction strategies for the periplasmic production of recombinant human IFN-α2b (interferon-α2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl β-D-thiogalactoside) concentration and induction temperature. Secondorder polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-α2b formation and percentage of soluble IFN-α2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D 600 ), IPTG concentration of 0.05 mM and temperature of 25 • C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-α2b in the periplasmic space with translocation of 74 % of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-α2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4fold respectively.
2010
Two plasmids, pFLAG-ATS and pET 26b(+), were studied for the periplasmic expression of recombinant human interferon-α2b (IFN-α2b) in Escherichia coli. The pFLAG-ATS contains ompA signal sequence and tac promoter while pET 26b(+) contains pelB signal sequence and T7lac promoter. It was observed that periplasmic expression of IFN-α2b from pET 26b(+) was around 3000 times higher than pFLAG-ATS. Difference in the expression level was attributed to the difference in the promoters and the signal sequences. In silico analysis of mRNA secondary structures were analyzed using Vienna RNA package and MFOLD. The results suggested that the increase of expression would mainly due to the difference in the translation initiation associated with secondary structure of mRNA transcribed by both plasmids.
The influence of different carbon and nitrogen sources on growth of recombinant Escherichia coli and human interferon-α2b (IFN-α2b) production in periplasmic space was studied in shake flask culture. A statistical method based on Plackett-Burman design was used to screen the main medium components that greatly influenced the performance of the fermentation process. The optimization of medium was performed using response surface methodology (RSM) where three critical factors (glucose, yeast extract and peptone) were optimized using central composite design. The highest yield of periplasmic recombinant human interferon-α2b (PrIFN-α2b) (335.8 µg/L) was predicted to be obtained in optimized medium containing 5.47 g/L glucose, 55.24 g/L yeast extract and 42.27 g/L peptone.. The production of IFN-α2b in periplasmic space in optimized medium was about 2.5, 11.7 and 124.4 times higher than Terrific broth (TB), Luria-Bertani (LB), and minimal medium (M9), respectively.
2009
The influence of different carbon and nitrogen sources on growth of recombinant Escherichia coli and human interferon-α2b (IFN-α2b) production in periplasmic space was studied in shake flask culture. A statistical method based on Plackett-Burman design was used to screen the main medium components that greatly influenced the performance of the fermentation process. The optimization of medium was performed using response surface methodology (RSM) where three critical factors (glucose, yeast extract and peptone) were optimized using central composite design. The highest yield of periplasmic recombinant human interferon-α2b (PrIFN-α2b) (335.8 μg/L) was predicted to be obtained in optimized medium containing 5.47 g/L glucose, 55.24 g/L yeast extract and 42.27 g/L peptone.. The production of IFN-α2b in periplasmic space in optimized medium was about 2.5, 11.7 and 124.4 times higher than Terrific broth (TB), Luria-Bertani (LB), and minimal medium (M9), respectively.
Biochemical Journal, 1994
Human interferon-a2c (IFN-a2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable entertoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-a2c could be increased from 25 % to more than 50 % by shifting the fermentation pH from 7.0 to 6.7.
2014
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and d...
Cloning and Expression of an Optimized Interferon Alpha 2b in Escherichia coli strain BL21 (DE3)
Annals of Tropical Medicine and Public Health, 2019
Interferon alpha 2b gene (INF α2b) as a protein with antiviral and antitumor activities is potentially a valuable therapeutic proteins to work on. Prior to a large scale production of the target protein, it is recommended to examine it in an experimental scale, so that bacterial host could be a proper choice as it leads us to a deep insight of the subject. In this research, INF α2b sequence obtained from NCBI gene data bank, and after optimization it was subjected to be cloned and expressed in pET28a+. In order to primary examination of the target protein, Escherichia coli was considered as a prokaryotic expression system. IPTG induction of the protein in bacteria cells containing the construct pET: IFN, followed by resolving total proteins through SDS-PAGE. The expected size of the investigated protein, about 24kDa, observed through gel separation. Further assessment via western blotting confirmed successful expression of IFN α2b.
Maximizing Production of Human Interferon-γ in HCDC of Recombinant E. coli
Iranian journal of pharmaceutical research : IJPR, 2013
Tuning recombinant protein expression is an approach which can be successfully employed for increasing the yield of recombinant protein production in high cell density cultures. On the other hand, most of the previous results reported the optimization induction conditions during batch and continuous culture of recombinant E. coli, and consequently fed-batch culture have received less attention. Hence, in this research induction conditions for the over-production of recombinant interferon-γ including the amount of inducer, induction time and post-induction duration during chemical induction were optimized. E. coli BL21 (DE3) (pET3a-hifnγ) was used to over-express human interferon-gamma (hIFN-γ) in an exponential fed-batch procedure with a maximum attainable specific growth rate of 0.55 h(-1) at the beginning of feeding and 0.4 h(-1) in induction time. The factors were considered as the amount of inducer (IPTG) in the range of 0.565-22 mg g(-1) L(-1) at seven levels, cell density at i...
In the present work, a recombinant Escherichia coli strain was used for the production of interferon α-2b in both shake flask and in bioreactor. The first part of this research was focused on the investigation of the effect of glucose concentration on the kinetics of cell growth, recombinant protein production and acetate formation. In general, glucose supplementation to culture medium enhanced cell growth when added in concentration between 0-20 g/L. Further increase in glucose level reduces biomass production and enhances acetate accumulation in culture. The results clearly demonstrated that maximal interferon production of 27.7 mg/L was achieved in culture supplemented with 20 g/L glucose. Further improvement in recombinant interferon production process was also achieved by scaling up from shake flask to 16-L stirred tank bioreactor. The maximal volumetric interferon production in bioreactor batch culture was 44.5. mg/L after only 6 hours.
Enhancement of Human γ-Interferon Production in Recombinant E. coli Using Batch Cultivation
Applied Biochemistry and Biotechnology, 2010
Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at latelog phase (OD 600 =4.5). Under these conditions, dry cell weight of 6±0.2 g/l and γ-interferon concentration of 2.15±0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14±0.2 g/l, concentration and overall productivity of γ-interferon 4.2±0.1 g/l and 420±10 mg/l h, respectively, were obtained.