Production Of Human Interferon Gamma Research Articles

Fusion to elastin-like polypeptide increases production of bioactive human IFN-γ in tobacco.

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The ELP fusion proteins were successfully recovered from total soluble protein with adding 2.75M NaCl and three rounds of inverse transition cycling (ITC). The hIFN-γ was also separated from ELP with Enterokinase cleavage of the fusion protein and recovered by ITC. Inverse transition analysis indicated that the hIFN-γ-ELP variants aggregate above their inverse transition temperature and at high ionic strength. Investigation of glycosylation revealed that fused or unfused hIFN-γ proteins are N-glycosylated in different cellular locations. Moreover, N-glycosylation analysis and bioassay showed that fusion to ELP does not disturb glycosylation process and antiviral activity of hIFN-γ.

Artificial neural network‐genetic algorithm (ANN‐GA) based medium optimization for the production of human interferon gamma (hIFN‐γ) in Kluyveromyces lactis cell factory

AbstractIn the current investigation, we have adapted response surface methodology (RSM) and artificial neural network‐genetic algorithm (ANN‐GA) based optimization to develop a defined medium for maximizing human interferon gamma production from recombinant Kluyveromyces lactis (K. lactis). In the initial screening studies, sorbitol and glycine emerged as a carbon and nitrogen source respectively having higher influence on hIFN‐γ production. Substrate inhibition studies were performed by varying the initial substrate concentration, and we found maximum hIFN‐γ concentration at 50 g L−1 of sorbitol. Inhibition kinetics studies were carried out using 3 and 4‐parametric models. Among the estimated models, the Moser model was observed as the best fitted model followed by the Luong model with R2 values of 0.882 and 0.75, respectively. The model acceptability test was carried out using the extra sum of squares F‐test and Akaike information criterion (AIC). The Plackett‐Burman multifactorial design identified sorbitol, glycine, Na2HPO4, and MgSO4.7H2O as the parameters significantly influencing the hIFN‐γ production. Further, the Box‐Behnken design (BBD) followed by the artificial neural network coupled with genetic algorithm (ANN‐GA) was employed for the precise optimization of medium components. With ANN‐GA a maximum hIFN‐γ yield of 2.1 ±0.3 mg L−1 in shake flask level and 3.5 ±0.1 mg L−1 in reactor level was achieved. The findings of this study serve as a model for a process development strategy (bench scale to reactor scale) to achieve a high productivity of the desired protein from a microbial cell factory.

Genetic and substrate-level modulation of Bacillus subtilis physiology for enhanced extracellular human interferon gamma production

ABSTRACTHuman interferon-gamma (hIFNG) production is limited by various gene-level bottlenecks including translation, protein folding, and secretion which depends upon the physiological state of the organism. In this study gene-level and substrate-level modulations have been used to control Bacillus subtilis physiology for >15 fold extracellular soluble hIFNG production. Two variants of the native human interferon-gamma gene (hifng) were designed and synthesized, namely, cohifnghis and cohifng having codon adaptation index 25.33 and 26.89% higher than the native gene, respectively. BScoIFNG and BScoIFNGhis with ΔG of −100.0 and −113.7 kcal mol−1 resulted in 30 and 6.5% higher hIFNG compared to the native gene in complex medium. BScoIFNG produced 1.53 fold higher hIFNG using glucose-based defined medium as compared to the complex medium by modulating the physiological parameter growth rate from 0.35 to 0.26 hr−1. Further modulatory effect of various phosphotransferase transport system (PTS) and no-PTS sugars, sugar alcohols, and organic acids was quantified on the physiology of B. subtilis WB800N for extracellular hIFNG production. Sorbitol and glycerol emerged as the best hIFNG producers with lowest growth and substrate consumption rates. BScoIFNG produced maximum 3.15 mg L−1 hIFNG at 50 g L−1 glycerol with highest hIFNG yield (Yp/x = 0.136) and lowest substrate uptake rate (qs = 0.26).

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

ABSTRACTThe present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80 g/L lactose, 10.353 g/L yeast extract, and 15 mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881 µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1 mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.

Improving the production of human interferon gamma (hIFN-γ) in Pichia pastoris cell factory: An approach of cell level

Human interferon gamma (hIFN-γ) has an antiviral, immunoregulatory and anti-tumor properties. It is majorly used for the treatment of chronic granulomatous disease and severe malignant osteopetrosis. In the present investigation, cell level strategies were applied to address the expression machinery related problems, which are bottle neck for protein expression in Pichia pastoris. The gene encoding hIFN-γ was successfully cloned and expressed in P. pastoris (GS115) under the control of AOX promoter. About 200μg/L of recombinant human interferon gamma (rhIFN-γ) was expressed after 1% methanol induction. The expression of rhIFN-γ was further enhanced by 2.67 fold by co-expressing the gene encoding protein disulphide isomerase (PDI) along with hIFN-γ. Furthermore, the effect of codon bias was examined by cloning codon optimized hIFN-γ in P. pastoris (GS115) that resulted in about 9 fold enhancement of rhIFN-γ production. Optimization of process parameters viz., temperature, pH, methanol concentration, inoculum size and agitation rate enhanced the production of rhIFN-γ by 12.5 fold. The purification with IMAC (Immobilized metal ion affinity chromatography) showed about 56.5, 63.83 and 80% purity of rhIFN-γ from GS-hIFN-γ, GS-hIFN-γ-PDI and GS-hIFN-γopt strains, respectively.

Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis

Integrative and replicative plasmids for the expression driven by the P 43 promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide ( sp-amyL) encoding sequence. The integrative construct produced 2.5 ± 0.2 mg l −1 and the replicative system produced 20.3 ± 0.8 mg l −1 of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.

Optimization of human interferon gamma production in Escherichia coli by response surface methodology

The production of human interferon gamma (hIFN-γ) using a synthetic gene in Escherichia coli BL21-SI was optimized by response surface methodology (RSM) and a Box-Behnken design. The process variables studied were temperature, bio-mass concentration at induction time and the NaCl concentration as inducer. According to the Box-Behnken design, a second order response function was developed. The optimal expression conditions were a temperature of 32.6°C, induction biomass of 0.31 g/L and 0.3 M NaCl in minimal medium. The model prediction for the maximum hIFN-γ production was 77.3 mg/L, which corresponded satisfactorily with the experimental data. The hIFN-γ concentration attained under optimized conditions was 13-times higher than that obtained using the non-optimized conditions. We conclude that RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Platelet-activating factor and the production of human interferon-gamma.

Platelet-activating factor (PAF) was tested for its ability to alter yields of human interferon (IFN) produced from peripheral blood mononuclear cells (PBMC). Using different concentrations of phytohemagglutinin (PHA) we could not demonstrate a consistent effect of PAF at any concentration tested on the yield of IFN-gamma. Similarly, although PAF was associated with a slight enhancement of IFN-gamma yields when PBMC were induced by interleukin-2 (IL-2), the results were not statistically significant. No effect was observed on the accumulation of human IFN-gamma mRNA induced by PHA. Furthermore, PAF did not enhance yields of IFN-gamma when the cells were induced by poly I:poly C. We conclude that although PAF may have a role in sepsis, it is not likely that this is in any way related to its ability to significantly alter the yield of interferons.

Potent inhibition of both human interferon-gamma production and biologic activity by the Clara cell protein CC16.

The Clara cell 16 kD protein (CC16), the predominant product of the Clara cells lining the bronchiolar epithelium, is thought to protect the respiratory and urogenital tract from unwanted inflammatory reactions through its immunosuppressive action. In this report, we show evidence that CC16 establishes an anti-inflammatory activity by interfering with the interferon-gamma (IFN-gamma)-mediated actions of the cytokine network. The HuIFN-gamma production of stimulated single-donor peripheral blood mononuclear cells is inhibited by the presence of doses of CC16 in the range of 10(-12) M, with a maximal inhibition (up to 95%) when interleukin-2 is used as a stimulating agent. CC16 also diminishes the biologic activity of IFN-gamma: both the antiviral activity and the stimulation of phagocytosis by IFN-gamma, measured by means of chemiluminescence, are reduced in the presence of CC16. These observations indicate that CC16 acts as an anticytokine and could give new insight in the potential role of the Clara cells.

Reversal of the effects of cyclosporin A on the production of human interferon-gamma.

Previous reports have documented that cyclosporin A (CsA) can inhibit the production of interferon-gamma (HuIFN-gamma) and interleukin-2 (IL-2). In this report we have extended these earlier findings and have demonstrated that the defect in the production of HuIFN-gamma persists even after the CsA is removed and is associated with altered synthesis of other lymphokines, mainly a cytotoxin and leukocyte migration inhibition factor. In the presence of recombinant exogenous IL-2, the effect of CsA on the synthesis of HuIFN-gamma can be reversed depending on the concentrations of both the IL-2 and CsA. In addition, the presence of the calcium ionophore A23187 partially reverses the inhibitory effect of CsA by synergistically enhancing the synthesis of HuIFN-gamma.

Production of human interferon-gamma in serum-free medium.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.