Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product

Retnoningrum, Debbie Soefie; Ningrum, Ratih Asmana; Kurniawan, Yohanes Novi; Indrayati, Ana; Rachmawati, Heni

doi:10.1016/j.jbiotec.2009.11.008

Cited by 13 publications

(14 citation statements)

References 12 publications

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“…Retnoningrum et al constructed a synthetic open reading frame encoding human IFN α 2b for over expression as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFN α 2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified with a yield of 3.46 mg/L [21]. Valente et al used E. coli codon optimized artificial gene that produced 16.6 mg/L of IFN α 2b [22].…”

Section: Discussionmentioning

confidence: 99%

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Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

Jin

Huang

et al. 2014

BioMed Research International

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Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

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Section: Discussionmentioning

confidence: 99%

“…IFN α 2b is the first one used in the clinic for the treatment of chronic hepatitis B and C and several cancers such as melanoma, AIDS-related Kaposi's sarcoma, chronic myeloid lymphoma, and angioblastoma [3]. However, because interferon does not have organ-specific affinity, patients may not always achieve a therapeutic effect [4].…”

Section: Introductionmentioning

confidence: 99%

Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

Jin

Huang

et al. 2014

BioMed Research International

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“…For more than a decade, interferon therapy is the gold standard in treatment for certain forms of viral hepatitis and carcinogenesis [9]. However, therapeutic efficacy has been limited because interferon does not have organ-specific affinity and its in vivo half-life is short [10].…”

Section: Introductionmentioning

confidence: 99%

High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo

Wang

Jin

et al. 2015

BMC Biotechnol

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BackgroundIn our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide was successfully constructed. IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells. The aim of the present investigation was focused on how to produce high levels of recombinant IFN-CSP and its in vivo anti-hepatitis B virus (HBV) activity.MethodsA modified DNA fragment encoding IFN-CSP was synthesized according to Escherichia coli (E. coli) preferred codon usage and transformed into E. coli BL21 (DE3) for protein expression. The induction conditions were systematically examined by combining one-factor experiments with an orthogonal test (L(9)(3)(4)). The antigenicity of the purified protein was characterized by western blot analysis. The in vivo tissue distribution were assayed and compared with native IFN α2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.ResultsThe results showed that the E. coli expression system was very efficient to produce target protein.ConclusionOur current research demonstrates for the first time that IFN-CSP gene can be expressed at high levels in E. coli through codon and expression conditions optimization. The purified recombinant IFN-CSP showed liver-targeting potentiality and anti-HBV activity in vivo. The present study further supported the application of IFN-CSP in liver-targeting anti-HBV medicines.

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“…The amino acid sequence of human interferon alpha 2b (hIFNα-2b) is highly similar to those of interferon alpha 2a (hIFNα-2a) with a difference of only one amino acid at position 23 (arginine in case of hIFNα-2b and lysine in case of hIFNα-2a (Retnoningrum et al 2010). …”

Section: Introductionmentioning

confidence: 99%

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b

et al. 2013

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Interferon alpha 2b (IFNα-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFNα-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFNα-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFNα-2b is 19 kDa. The over expressed recombinant hIFNα-2b was checked by ELISA using antibodies raised against commercially available hIFNα-2b. The biocomputational analysis of recombinant hIFNα-2b gene showed the 99.9% nucleotide sequence and 100% deduced amino acid sequence homology with reported sequences of IFNα-2b. The predicted 3D-structure showed mainly five α-helices, one 310 helix and two disulfide bonds at Cys1-Cys98 and Cys129-Cys138. The amino acid sequence alignment indicated that the disulfide linkage position is conserved in all IFNα family members. On the basis of sequence homology among interferon alpha family, new potent variants of hIFNα-2b with enhance efficacy can be produced. Indigenous production of IFNα-2b from gene of local population will reduce the cost and increase tolerability of interferon therapy.

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Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product

Cited by 13 publications

References 12 publications

Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b

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