EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN CONSENSUS INTERFERON-ALPHA IN<i>Escherichia coli</i>UNDER λP<sub>L</sub>PROMOTER

Mohammed, Yasir I.; El-Baky, Nawal Abd; Redwan, Elrashdy M.

doi:10.1080/10826068.2011.637600

Cited by 19 publications

(23 citation statements)

References 24 publications

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“…Cytotoxicity effect of purified camel IgGs, α-lactalbumin, cLf, casein, or IVIGs on human separated PBMCs and Huh7.5 cells was tested by Thiazolyl Blue Tetrazolium Bromide (MTT) assay [49-51] PBMCs cells and Huh7.5 cells (10 4 ) were cultured in 96-well microtiter plates in duplicate and cultured overnight at 37 °C, then the medium was refreshed with DMEM supplemented medium containing 1.0 or 2.0 mg/ml of IgGs, α-lactalbumin, cLf, casein, or IVIGs. Cells treated with proteins were incubated for 4 days at 37 °C, 5 % CO 2 and 88 % humidity.…”

Section: Methodsmentioning

confidence: 99%

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Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

El‐Fakharany

Abedelbaky

Haroun

et al. 2012

Virol J

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PurposeTo extend the study of the camel milk proteins which have antiviral activity against HCV, camel naïve polyclonal IgGs, α-lactalbumin were purified from camel milk and their anti-HCV effect was examined using PBMCs and Huh7.5 cell-lines. They were compared with the activity of human polyclonal IgGs and camel lactoferrin and casein.Material and methodsThree types of experiments were performed on PBMCs and HuH7.5 cell. HCV was directly incubated with the purified proteins and then mixed with both cell types, or the proteins were incubated with the cells and then exposed to HCV, or the HCV pre-infected cells were treated with the proteins to inhibit intracellular replication. The proteins were added to cells or virus at different concentrations and time intervals.ResultsPretreated PBMCs and Huh7.5 cells with milk proteins were not protected when exposed to HCV infection. The direct interaction between HCV and camel IgGs and camel lactoferrin (cLf) led to a complete inhibition of HCV entry into cells, while casein, α-lactalbumin and human IgGs failed to inhibit HCV entry at any tested concentration. Camel IgGs showed ability to recognize HCV peptides with a significant titer (12 × 103) in comparison with human IgGs which failed to do it. Camel lactoferrin was capable of inhibiting the intracellular HCV replication at concentrations of 0.25-1.25 mg/ml.ConclusionCamel milk naïve polyclonal IgGs isolated from camel milk could inhibit the HCV infectivity and demonstrated strong signal against its synthetic peptides. Lactoferrin inhibit the HCV infectivity started from 0.25 mg/ml. However, α-lactalbumin, human IgGs and casein failed to demonstrate any activity against HCV infectivity.

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Section: Methodsmentioning

confidence: 99%

“…Then, it was incubated at 37 °C, 5 % CO 2 and 88 % humidity for 5 hours to allow the MTT to be metabolized. Finally, 200 μl of dimethylsulfoxide were added to each well, placed on the shaking table at 150 rpm for 5 min and the optical density was read at 595 nm [49-51]. …”

Section: Methodsmentioning

confidence: 99%

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

El‐Fakharany

Abedelbaky

Haroun

et al. 2012

Virol J

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“…They achieved a yield of 5.5 g/L interferon at a cell density of 68 g of dry cell weight (DCW)/L but maintaining two reactors may have restricted the use of this design. We have previously reported heat-inducible expression of cIFN-α protein in E. coli with a λP L promoter system [ 13 ]. Most of the expressed protein aggregated into inclusion bodies, therefore needed in vitro refolding.…”

Section: Introductionmentioning

confidence: 99%

Auto-induction expression of human consensus interferon-alpha in Escherichia coli

2015

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BackgroundIsopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a.ResultsThe expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a.ConclusionsThe auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production.

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“…Egyptians are predominantly infected with HCV genotype 4 (subtype 4a), which has a reduced response rate to standard interferon (IFN)-based therapy compared with genotypes 1, 2, or 3 (54). The standard therapy for Egyptian HCV patients is the use of IFN as a monotherapy (IFN-α2a, -α2b and pegylated IFN) (55,56) or combined with ribavirin; however, patients often use various alternative medicines (57–59). Recent studies investigated the use of whey protein concentrate (57), pasteurized camel milk (58) and fresh camel milk (59) in the treatment of HCV genotype 4 infection; the results demonstrated significant decreases in viral load, markers of active inflammation and ALT and AST activity (57–59) following treatment.…”

Section: Discussionmentioning

confidence: 99%

Influence of camel milk on the hepatitis C virus burden of infected patients

El‐Fakharany

El-Baky

Linjawi

et al. 2017

Experimental and Therapeutic Medicine

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Hepatitis C virus (HCV) infection represents a world health problem and no protective vaccine or effective drug currently exists. For economic reasons, many patients use traditional medicines to control the infection. In Egypt, camel milk is one of the traditional medicines widely consumed by patients infected with HCV. The present study aimed to evaluate the efficacy of camel milk in the treatment of patients infected with HCV. Whole camel milk from a local farm was administered to patients for 4 months (250 ml/day/patient). Patient sera were collected prior to and following camel milk drinking, and three markers were set-up for sera-evaluation. The three markers indicating the effect of camel milk on HCV infection were: Liver function assays [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)]; a viral load assay; and anti-HCV antibodies profile and isotyping against synthetic HCV epitopes. Camel milk demonstrated the ability to improve general fatigue, health and liver function (ALT and AST levels); ALT was reduced in ~88% of patients and AST was reduced in all patients subsequent to drinking camel milk for four months. The majority of patients responded positively to camel milk treatment; RNA viral load decreased in 13 out of the 17 patients (76.47%) and one patient exhibited undetected viremia following camel milk treatment. The anti-HCV antibodies profile and isotyping were significantly decreased (P<0.05) in immunoglobulin (Ig)G1 following treatment in 70–76% of patients. However, the treatment was ineffective in 23.53% of patients who experienced no reduction in RNA viral load following treatment with camel milk. In conclusion, whole camel milk treatment demonstrated efficacy in vivo; the viral load in the majority of patient sera was reduced and the IgG isotype profile was converted to Th1 immunity.

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EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN CONSENSUS INTERFERON-ALPHA INEscherichia coliUNDER λP_LPROMOTER

Cited by 19 publications

References 24 publications

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

Auto-induction expression of human consensus interferon-alpha in Escherichia coli

Influence of camel milk on the hepatitis C virus burden of infected patients

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EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN CONSENSUS INTERFERON-ALPHA INEscherichia coliUNDER λPLPROMOTER

Cited by 19 publications

References 24 publications

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

Auto-induction expression of human consensus interferon-alpha in Escherichia coli

Influence of camel milk on the hepatitis C virus burden of infected patients

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EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN CONSENSUS INTERFERON-ALPHA INEscherichia coliUNDER λP_LPROMOTER