Induction Strategies for Interferon-α2B Production in Periplasmic Space of Escherichia Coli

Interferon-α2b (IFN-α2b) is a member of cytokine family with the ability to induce antiproliferative, antiviral, antineoplastic and immunomodulating activities. It is frequently used in the treatments of gastrointestinal tract diseases, various cancers and chronic hepatitis B. Due to its diagnostic and therapeutic potentials the production of IFN-α2b in large quantities is required to meet the demands from research, clinical and industrial applications. This study was carried out to enhance the production of recombinant IFN-α2b in the periplasmic space of E. coli using the recombinant E. coli Rosetta-gami 2(DE3) harboring the plasmid pET26b-IFN-α2b. In this study, different induction parameters (temperature, IPTG concentration, point of induction and post induction time) were analyzed and optimized during induction for optimal periplasmic IFN-α2b production. Further analysis was done on the effect of pH medium on IFN-α2b production in different fractions. Preliminary screening performed on four induction parameters shows that only three parameters (temperature, IPTG concentration and point of induction) improved the IFN-α2b production. These parameters were optimized individually by narrowing the working range as follows: temperature from 16°C to 30°C, IPTG lower than 2 mM, and induction point between early-log phase and mid-log phase on the growth curve of E. coli. A response surface methodology (RSM) based on a central composite design was used to optimize the induction parameters. The proposed induction strategy consisted of three optimized parameters: i) induction point at A600 of 4, ii) IPTG strength at 0.05 mM, and iii) induction temperature at 25°C. The strategy yielded 1.21 μg/mL of IFN-α2b, which represents 82% of the soluble IFN-α2b that was successfully transferred to the periplasmic space of E. coli after 18 h of induction. A study on the effect of pH on IFN-α2b production in different fractions reveals that the highest yield was obtained at pH 7 with a high buffering capacity (0.2 M). At this pH, a higher IFN-α2b production was obtained in the periplasmic fraction. Whereas, the amount of inclusion bodies formed was reduced. This was evident by the lack of aggregated materials in the cytoplasm of E. coli observed under the electron microscope. This study also proved that the suitable buffering capacity would maintain the pH of media during fermentation. Finally, when the optimized induction parameters with the appropriate media pH was applied, the total IFN-α2b obtained was 1.43 μg/mL, a 86.9-fold increase in productivity compared to the IFN-α2b produced under non-optimal condition (16.26ng/mL). Thus, proper optimization of fermentation conditions was proved useful to improve the production of periplasmic IFN-α2b in E. coli

MORPHOLOGICAL, BIOCHEMICAL AND TRANSCRIPTOMIC CHARACTERISATION OF Chlorella sorokiniana AND Chlorella zofingiensis DURING NORMAL AND STRESS CONDITIONS

Chlorella has been identified as one of the most interesting microalgae species, which has high nutritional values, high growth rate, and is able to produce a wide range of metabolites in response to environmental changes. The objectives of this study are to characterise the morphology and biochemical contents and to identify the genes and miRNAs involved in regulating the production of carotenoids and lipids in Chlorella sorokiniana and Chlorella zofingiensis when cultured under high light intensity combined with glucose supplementation. In this study, stress was introduced to the Chlorella cultures by adding 2% glucose and increasing the light intensity from 10 to 100 µmol photons m-1 s-1. Then, the pigments, total phenolic contents, and antioxidant activities of both Chlorella species were evaluated. The results showed that both strains grew larger when cultured under stress condition. Although the total carotenoid content was increased under stress condition, reduction of the pigment and total phenolic contents associated with lower antioxidant activity were also recorded. Subsequently, the transcriptome of C. sorokiniana was sequenced using Illumina paired-end sequencing, and 198,844,110 raw reads with the length of 100 bp were produced. After pre-processing, ~95% of high quality reads were de novo assembled using Trinity software into 18,310 contigs. Analysis of differential gene expression by DESeq2 package showed that a total of 767 genes were upregulated and 948 genes were downregulated in stress conditions. Then, miRNAs that regulate the genes during normal and stress conditions of both C. sorokiniana and C. zofingiensis were profiled and analysed using CLC Genomic Workbench and OmiRas. From both analysis pipelines, the known and predicted novel miRNAs were identified. Although most of the identified miRNAs were not functionally determined, this study suggests that they were species-specific, which may have roles in regulating genes during stress condition. In conclusion, identifying the genes and the regulation of various metabolite productions under different growth conditions are useful for further strain enhancement of the microalgae

Optimization of an induction strategy for improving interferon-α2b production in the periplasm of Escherichia coli using response surface methodology

Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) 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 beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b 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 D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b 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-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively

A comparison of the morphological and biochemical characteristics of Chlorella sorokiniana and Chlorella zofingiensis cultured under photoautotrophic and mixotrophic conditions

The responses of two species of microalgae, Chlorella sorokiniana and Chlorella zofingiensis, were compared regarding their morphological and biochemical properties under photoautotrophic and mixotrophic conditions. These microalgae were cultured under both conditions, and their crude ethanolic extracts were examined for their pigment and total phenolic contents. In addition, the microalgae's antioxidant activities were determined using a DPPH radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Both strains showed increases in cell size due to the accumulation of lipid bodies and other cell contents, especially carotenoids, under the mixotrophic condition. Notably, reductions in phenolic and chlorophyll contents were observed to be associated with lower antioxidant activity. C. zofingiensis compared with C. sorokiniana, demonstrated higher antioxidant activity and carotenoid content. This study showed that different species of microalgae responded differently to varying conditions by producing different types of metabolites, as evidenced by the production of higher levels of phenolic compounds under the photoautotrophic condition and the production of the same levels of carotenoids under both photoautotrophic and mixotrophic conditions

Enhanced interferon-α2b production in periplasmic space of Escherichia coli through medium optimization using response surface method

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

Identification and analysis of microRNAs in Chlorella sorokiniana using high-throughput sequencing

Chlorella is a popular microalga with robust physiological and biochemical characteristics, which can be cultured under various conditions. The exploration of the small RNA content of Chlorella could improve strategies for the enhancement of metabolite production from this microalga. In this study, stress was introduced to the Chlorella sorokiniana culture to produce high-value metabolites such as carotenoids and phenolic content. The small RNA transcriptome of C. sorokiniana was sequenced, focusing on microRNA (miRNA) content. From the analysis, 98 miRNAs were identified in cultures subjected to normal and stress conditions. The functional analysis result showed that the miRNA targets found were most often involved in the biosynthesis of secondary metabolites, followed by protein metabolism, cell cycle, and porphyrin and chlorophyll metabolism. Furthermore, the biosynthesis of secondary metabolites such as carotenoids, terpenoids, and lipids was found mostly in stress conditions. These results may help to improve our understanding of regulatory mechanisms of miRNA in the biological and metabolic process of Chlorella species. It is important and timely to determine the true potential of this microalga species and to support the potential for genetic engineering of microalgae as they receive increasing focus for their development as an alternative source of biofuel, food, and health supplements

Effect of promoter strength and signal sequence on the periplasmic expression of human interferon-α2b in Escherichia coli

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 resultssuggested 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

Improvement of foundation student performance in biology through intensive station based learning approach

A growing revolution is underway in teaching introductory science to foundation studies. Recent educational research explains that traditional teaching approaches in large classes often fail to reach many students. To address this problem, we conducted an intensive station rotation-based workshop called “Bio Made Easy” for a group of students who obtained F grade in the first Biology assessment, N = 120. The workshop was designed to improve students’ understanding of selected Biology topics by providing simple examples, analogy, and explanation of the concept using various active and interactive approaches. The goal was to change the students’ perception of biology and deliver the content within a short period of time. The students were divided into small groups and required to complete all stations conducted by different instructors. For each station, students were directed to perform activities that required them to actively participate, interact, and discuss among the group members. A comparison was made between their performance during the first test and the second test of the semester. From F grade in the first test, about 88.3% of the students accomplished higher performance grades in the test 2 assessment. This reflects an encouraging sign that active-learning practice and direct engagement of students in the station rotation-based learning approach improve student performance in biology subject and serve as one of the strategies to motivate students for better grades

A comparison of the morphological and biochemical characteristics of Chlorella sorokiniana and Chlorella zofingiensis cultured under photoautotrophic and mixotrophic conditions

The responses of two species of microalgae, Chlorella sorokiniana and Chlorella zofingiensis, were compared regarding their morphological and biochemical properties under photoautotrophic and mixotrophic conditions. These microalgae were cultured under both conditions, and their crude ethanolic extracts were examined for their pigment and total phenolic contents. In addition, the microalgae’s antioxidant activities were determined using a DPPH radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Both strains showed increases in cell size due to the accumulation of lipid bodies and other cell contents, especially carotenoids, under the mixotrophic condition. Notably, reductions in phenolic and chlorophyll contents were observed to be associated with lower antioxidant activity. C. zofingiensis compared with C. sorokiniana, demonstrated higher antioxidant activity and carotenoid content. This study showed that different species of microalgae responded differently to varying conditions by producing different types of metabolites, as evidenced by the production of higher levels of phenolic compounds under the photoautotrophic condition and the production of the same levels of carotenoids under both photoautotrophic and mixotrophic conditions

Screening for the optimal induction parameters for periplasmic producing interferon-α2b in Escherichia coli

Screening for optimum induction parameters to improve the production of periplasmic interferon-α2b (PrIFN-α2b) by recombinant Escherichia coli was conducted using shake flask culture. Recombinant E. coli Rosetta-gami 2(DE3) harboring the plasmid pET26b containing IFN-α2b gene under the control of the T7lac promoter was used, where the induction was accomplished by isopropyl β-D-1-thiogalactopyranoside (IPTG). The induction parameters (inducer concentration, point of induction, induction temperature and the length of induction) were analyzed to find the suitable range to be used for further optimization process. From the analysis, narrow range of induction temperature from 16 to 30°C and IPTG lower than 2 mM were found suitable for induction of PrIFN-α2b. On the other hand, early log phase was the preferred time to initiate the induction and the length of induction was dependent on the combination of other induction parameters used