Production Of Human Interferon Research Articles

The Production of Soluble Human Gamma Interferon in the Escherichia coli Expression System with a Decrease in Cultivation Temperature

The Production of Soluble Human Gamma Interferon in the Escherichia coli Expression System with a Decrease in Cultivation Temperature

Removal of N-terminal methionine of human interferon α-2b by co‐producing with Pyrococcus furiosus methionine aminopeptidase in Escherichia coli

In our previous studies, expression of human interferon α-2b gene in Escherichia coli gave three types of polypeptide chains in nearly equimolar ratio. They include molecules with N-terminal methionine, molecules without N-terminal methionine, and molecules with acetylated N-terminal. This heterogeneity is not required in therapeutic applications. In order to improve the removal rate of N-terminus methionine, co-expression of human interferon α-2b and methionine aminopeptidase genes was performed in E. coli. The co-expression resulted in production of human interferon α-2b with dominance of molecules without N-terminal methionine and very few of them were acetylated at the N-terminus. Our findings contribute significantly to efforts for the production of homogenous therapeutic proteins which represent the exact recombinant replica of their native proteins.

코돈 최적화된 유전자를 이용한 재조합 대장균으로부터 인간 인터페론 베타 발현

The multiple sclerosis caused by multiple inflammatory disease or immune system disorder, is usually treated by interferon β through adjusting the abnormal immune reactions. For high production of human interferon β using recombinant E. coli, codon optimized and wild type genes were synthesized. When pET-15b or pET-21a vector was used as an expression vector with each gene, there was no target protein expression. When pQE30 vector was used as an expression vector, human interferon β was expressed by recombinant E. coli XL1-blue and E. coli JM109. Using the codon optimized gene, the expression of human interferon β was slightly increased as compared to that from wild type gene. However, most of expressed human interferon β was insoluble form.

The W-Shaped Mortality-Age Distribution of Novel H1N1 Influenza Virus Helps Reconstruct the Second Wave of Pandemic 1918 Spanish Flu

Interferon is essential in human defense against influenza virus. The non-structural gene segment (NS) of influenza virus has a critical role in counteracting human interferon-mediated antiviral responses. The second wave of 1918 H1N1 Spanish influenza pandemic was characterized by an enhanced mortality and a W-shaped mortality age distribution. In contrast to the U-shaped mortality-age distribution that targeted the very young and elderly during the first wave, young adult population were also affected during the second wave. The NS of the 1918 H1N1 Spanish influenza virus (1918PV) isolated during the second wave contributes to the virulence of 1918PV. This unique NS of 1918PV is able to inhibit human interferon production at both the pre-transcriptional and post-transcriptional level and induce cytokine dysregulation. The NS of 1918PV has entered the swine population in 1918 and re-emerged in the 2009 novel H1N1 influenza A pandemic virus (2009PV). Both seasonal and pandemic novel H1N1 influenza A viruses produced a W-shaped mortality age distribution. Information from the 2009 novel H1N1 Influenza A pandemic may help to reconstruct the mysterious surge in mortality during the second wave in the 1918 H1N1 Spanish influenza A pandemic. The W-shaped mortality-age distribution of 2009PV indicates the importance of a universal influenza vaccination policy for public protection. The high incidence of cytokine dysregulation and Streptococcus pneumoniae co-infection in hospitalized patients reflects the importance of pneumococcal vaccination and the development of immunomodulating agents that can control influenza-induced cytokine dysregulation.

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF HUMAN INTERFERON ALPHA2b GENE

Human alpha Interferons (hIFNα) have been shown to have antiviral, antiproliferative and immunomodulatory activities. The human interferon alpha2b (hIFNα2b), is one of the human interferon alpha2 sub variants, naturally synthesized as a polypeptide of 188 amino acid residues, the first 23 residues of which represents a signal peptide. In the present study, the hIFNα2b gene was expressed after being fused with Glutathione S-Transferase (GST) gene. The hIFNα2b gene was amplified from human genomic DNA by using a pair of specific primers, cloned into an Escherichia coli expression vector and expressed in E. coli cells under the direction of the tac promoter. The expressed protein was purified using a one-step affinity chromatography column containing immobilized gluthatione-bound resin. The purified protein was shown to react specifically with anti-human-interferon-alpha antibody, confirming that the protein was the human interferon alpha molecule. This strategy has the potential to be used as an alternative mean for production of pure human interferon α proteins for therapeutic purposes and for further studies on their molecular characterization and mechanism of action.

Nanofizyka ta antyvirusna terapiya

A new mechanism of interaction between viruses and nanoparticles is proposed. The mechanism is based on the local-field enhancement effect inherent only in nano-objects and can manifest itself in nanoparticle–virus systems. The basic idea consists in vacuum fluctuations that are always present in any physical system. This mechanism is universal and does not depend on the details of nanoparticle and virus structures, which was confirmed by numerous experiments carried out by us and in other scientific groups. A new method of purification of biofluids from nano-objects such as nanoparticles and viruses is also discussed. The method is based on a selective interaction between nano-objects and either a nanostructured surface, along which a surface plasmon-polariton propagates, or a system of nanothreads, on which a local plasmon-polariton is excited. On the basis of the method proposed for weakening the virus activity due to the action of a suspension of nanoparticles, a new effective way for the production of human leukocytic interferon has been developed and verified experimentally.

Large-scale production of human interferon by leukemic lymphoblasts grown in hamsters.

Large-scale production of human interferon by leukemic lymphoblasts grown in hamsters.

Expression of human interferon β in mammary glands of transgenic rabbits

A biotechnological system for the production of human beta interferon was developed on the basis of a hybrid gene constructed from the coding sequence of the beta interferon gene inserted into the first exon of the sheep beta lactoglobulin gene. It is intended for the expression of human beta interferon in mammary glands of transgenic animals. Two lines of transgenic rabbits were obtained using the hybrid gene. The tissue specificity of the expression of the transgene and the frequency of its inheritance in the first and second generations were studied. The activity of interferon was 2.2 x 10(4)-7.2 x 10(4) IU per milliliter of milk of transgenic female rabbits. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.

Highly efficient production of human interferon-.ALPHA. by transgenic cultured rice cells

Interferon-α (IFN-α) is an important antiviral pharmaceutical. A binary vector containing the first intron of the rice cytosolic SOD gene, the signal sequence of the 10 kDa rice prolamin, the amino-terminal region of β-glucuronidase, a thrombin recognition site, and the mature polypeptide region of human IFN-α was constructed, under the regulation of the cauliflower mosaic virus 35S promoter. Here, we report that transgenic rice cells transformed with this fusion protein vector produced a biologically active IFN-α. The vector was introduced into rice calli by Agrobacterium-mediated methods. Five lines of transgenic calli were obtained. IFN assay demonstrated that these calli expressed fusion proteins bearing biologically active IFN-α. Liquid-cultured cells exhibited stable growth and the production of active IFN-α during 10 successive generations, i.e. in 10 weeks. The expressed proteins were purified by immuno affinity chromatography and reverse-phase HPLC. Repeated selections of cultured cells that had been obtained by dividing calli into small cell aggregates considerably increased the production of IFN-α. Thrombin protease treatment of the fusion protein yielded the intact IFN-α polypeptide. Thus, transgenic suspension rice cells are expected to be useful for the production of large amounts of biologically active proteins at a low cost; moreover, such a system would be easier to employ than animal cell culture systems.

Gas-inducible product gene expression in bioreactors

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon- β (IFN- β ). Gaseous acetaldehyde-inducible IFN- β production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals.

Process development for production of recombinant human interferon-gamma expressed in Escherichia coli.

A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20-30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l(-1) after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

Изучение влияния различных концентраций индуктора на выход альфа-2b интерферона человека в системе экспрессии на основе РНК-полимеразы фага Т7 в клетках Escherichia coli

Изучение влияния различных концентраций индуктора на выход альфа-2b интерферона человека в системе экспрессии на основе РНК-полимеразы фага Т7 в клетках Escherichia coli

Изучение особенностей синтеза альфа-2b интерферона человека в клетках Escherichia coli при различных условиях культивирования

Изучение особенностей синтеза альфа-2b интерферона человека в клетках Escherichia coli при различных условиях культивирования

Metabolic flux analysis for human therapeutic protein productions and hypothesis for new therapeutical strategies in medicine

This work may be considered as a model study for therapeutic protein production, and a theoretical approach to hypothesise new medical strategies to further applied medical questions. A comprehensive generalised metabolic reaction network of Bacillus licheniformis that considers 149 reaction fluxes and 106 metabolites was used in the mass flux balance-based stoichiometric model for the analysis of human leukocyte interferon (IFN-α 1) and erythropoietin (EPO) production capacities of recombinant Bacillus species. The importance of cellular energetics on optimum performance was quantitatively assessed. The metabolic pathways leading to optimised IFN-α 1 and EPO overproduction were determined for the two carbon sources that have different reduction degrees ( γ), i.e. glucose ( γ=4.0) and citrate ( γ=3.0), and the variation of the fluxes were obtained. Metabolic capacity analyses showed that maximum IFN-α 1 and EPO synthesis rates were, respectively, 0.062 and 0.055 mmol g −1 DW h −1 at μ=0 h −1 when glucose uptake rate was 10 mmol g −1 DW h −1; and IFN-α 1 and EPO synthesis rates decreased, respectively, 1.70- and 1.75-fold when citrate was used as the carbon source. The flux distributions showed that the amino acid composition of the proteins influence the production. Leucine appears to be the most important amino acid for both IFN-α 1 and EPO production. Consequently, pyruvate seems to be the critical main branch point and B. pasteurii seems to be the favourable host for therapeutical protein production due to the high leucine uptake capacity. The results encourage the discussion on the potential strategies for improving production of IFN-α 1 and EPO, and further enable us to assert medical hypothesis in order to support the immune system of the human body against the deficiencies of the synthesis of IFN-α 1 and EPO in the human cells.

Candida albicans and Candida krusei differentially induce human blood mononuclear cell interleukin-12 and gamma interferon production.

Protection against Candida infection involves both innate and acquired immune responses, and cytokines produced by monocytes during the innate response may modify the acquired immune response by T cells. We hypothesized that Candida species which differ in pathogenicity can differentially induce production of immunoregulatory cytokines by human monocytes, which in turn modify T cells for immune responses to Candida. To test this hypothesis, we examined the effects of Candida albicans and Candida krusei on immunoregulatory cytokine production by human monocytes and gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC). Purified monocytes were incubated with live or heat-killed strains of C. albicans and C. krusei at the optimal Candida/monocyte ratio of 0.5. Cytokines in the supernatants were measured by enzyme-linked immunosorbent assay. Our data demonstrated that live C. albicans and C. krusei significantly induced interleukin-10 (IL-10), monocyte chemotactic factor 1, IL-1beta, and tumor necrosis factor alpha production by monocytes relative to unstimulated monocytes. In contrast, unlike C. krusei, pathogenic live strains of C. albicans induced no or only a minimal level of IL-12. The expression of IL-12 p40 mRNA levels by reverse transcription-PCR corroborated the IL-12 protein (p70) findings. In human PBMC, human blood monocytes were the major source of both IL-10 and IL-12 production in response to C. albicans and C. krusei. Upon activation of T cells in the presence of Candida-modified monocytes and antigen-presenting cells, IL-12 production by PBMC treated with Candida organisms correlated strongly with the level of IFN-gamma production by T cells. These results indicate that the virulence of C. albicans may be related to its ability to induce the monocytic type II cytokine IL-10, with a selective inhibition of IL-12 production, which may be responsible for the observed lack of T-cell IFN-gamma and may restrain an effective type I immune response to Candida.

Mild hyperthermia modulates biological activities of interferons

A significant enhancement of antiviral activity of human IFN-alpha, -beta and -gamma and murine IFN-gamma is observed when cells are treated with mild hyperthermia (39 degrees C) during antiviral assays. Treatment of primary human fibroblast cells with mild hyperthermia for 4 and 24 hours prior to interferon antiviral assays (pre-assay hyperthermia) further enhances interferon antiviral activity. An enhancement of interferon induced enzyme, 2,5-oligoadenylate synthetase, is also observed in cells treated with interferon and mild hyperthermia. This increase in enzyme activity is, in part, responsible for the observed increase in interferon antiviral activity with hyperthermia. Besides antiviral activity, mild hyperthermia also increases interferon antiproliferative activity on different tumour cells beyond its effect at normal physiological temperatures. On the other hand, mild hyperthermia decreases human interferon production in both human and murine cells when challenged with a viral or non-viral inducer. Also, mild hyperthermia suppresses interferon-mediated enhancement of natural killer (NK) cell activity in human and murine cells. The findings demonstrate that, although mild hyperthermia has suppressive effects upon interferon production and NK cell activity, it significantly increases both antiviral and antiproliferative activities of all three human interferons. These observations have direct bearing upon clinical utilization of exogenously administered interferons to late stage cancer patients who for the most part have a weaker immune system. In these patients, the antiviral and antiproliferative efficacies of administered interferon can be enhanced by combining interferon and hyperthermia.

Efficient production of human gamma interferon in tobacco protoplasts by genetically engineered brome mosaic virus RNAs.

We succeeded in producing human gamma interferon (IFN-gamma) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coat protein gene with the IFN-gamma gene and synthesized BMV-IFN-gamma chimera RNAs using an in vitro transcription system. The BMV-IFN-gamma chimera RNAs were used to inoculate tobacco protoplasts together with BMV RNA 1 and RNA 2 and produced IFN-gamma to a level of 5 to 10% of total extracted proteins per infected protoplast after 24 h of incubation. The efficient production of IFN-gamma was attributed to the high translation activity of the BMV-IFN-gamma chimera RNA. We demonstrate that 24 nucleotides coding for the N-terminal amino acids of the full-length coat protein were probably involved in the high translation activity of the BMV-IFN-gamma chimera RNA.

Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.

The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.

Production, purification and characterization of recombinant human interferon γ

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-γ (rhIFN-γ) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-γ obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-1 fermentor gave about 70 mg of a homogeneous preparation of rhIFN-γ. The specific biological activity of purified rhIFN-γ is ca. 3.4 · 10 7 I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (p I > pH 9) with a monomer relative molecular mass of 15 000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH 2-terminal sequence is identical with that established for the rhIFN-γ. However, its amino acid composition and its relative molecular mass (15 067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.

Production of recombinant human interferon- α1 by Escherichia coli using a computer-controlled cultivation process

Genetically engineered E. coli K12 BMH-71-18 with plasmid PBV-867 was used for constitutive expression of human interferon- α 1 (IFN) with a defined medium. A manual, time-based, fed-batch cultivation process produced a cell density of 26.3 g l −1 (OD 550 89), an IFN activity of 1.55 × 10 8 IU l −1 and a specific IFN productivity of 0.65 × 10 6 IU g −1. An analysis was conducted to characterize the problems involved in the high density microbial processes of recombinant protein production. The strategy suggested by the analysis is to establish a nutrient feeding profile that improves both the plasmid stability and the overall productivity of IFN. The nutrient feeding procedure developed here was based on the growth dynamics and a glucose consumption model. By using this procedure to continuously supply nutrients during cultivations, cell density reached 58 to 80 g l −1 and the specific IFN productivities of these runs were increased over that of the manual process. Nutrient feeding rates were found to affect the specific IFN productivity substantially. The optimized process achieved an IFN activity of 1.26 × 10 9 IU l −1, a cell density of 58 g l −1 and a specific IFN productivity of 2.2 × 10 7 IU g −1. More significantly, the overall productivity IU l −1 h −1 of the optimized, computer-controlled cultivation process was increased 12.9-fold over that of the manual cultivation process.