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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.

ABSTRACT: In our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN ?2b 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.A 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 ?2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.The results showed that the E. coli expression system was very efficient to produce target protein.Our 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.

SUBMITTER: Lu X

PROVIDER: S-EPMC4464711 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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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.

Lu Xuemei X Wang Jie J Jin Xiaobao X Zhu Jiayong J

BMC biotechnology 20150612

<h4>Background</h4>In 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.<h4>Methods</h4>A modified DNA fragment encoding IFN-CSP was synthesized according ...[more]

PMID: 26063245

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Project description:BACKGROUND:Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS:The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION:In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

Project description:BACKGROUND:Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. OBJECTIVES:The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. MATERIALS AND METHODS:The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-?-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS:The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37? for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. CONCLUSIONS:In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.

Dataset Information

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.

Publications

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.

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