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

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.

[1]  M. Levrero,et al.  IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. , 2012, The Journal of clinical investigation.

[2]  H. Mei,et al.  Expression of a novel dual-functional protein--the antimicrobial peptide LL-37 fused with human acidic fibroblast growth factor in Escherichia coli. , 2012, Protein expression and purification.

[3]  Yun Bai,et al.  An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples , 2011, Biotechnology Letters.

[4]  H. Mei,et al.  Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli , 2010, Applied Microbiology and Biotechnology.

[5]  Marina Etcheverrigaray,et al.  Highly glycosylated human alpha interferon: An insight into a new therapeutic candidate. , 2010, Journal of biotechnology.

[6]  R. A. Ningrum,et al.  Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product. , 2010, Journal of biotechnology.

[7]  S. Padmanabhan,et al.  Expression and Purification of SAK-fused Human Interferon Alpha in Escherichia coli , 2009 .

[8]  Y. Jeong,et al.  Superparamagnetic Iron Oxide Nanoparticles Coated with Galactose-Carrying Polymer for Hepatocyte Targeting , 2008, Journal of biomedicine & biotechnology.

[9]  E. Winzeler,et al.  Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite , 2007, Cell.

[10]  Prabhakar Tiwari,et al.  Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. , 2007, Molecular and cellular probes.

[11]  Amine Sadok,et al.  A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli. , 2007, Protein engineering, design & selection : PEDS.

[12]  Ana Rodriguez,et al.  The silent path to thousands of merozoites: the Plasmodium liver stage , 2006, Nature Reviews Microbiology.

[13]  T. McCutchan,et al.  An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion* , 2005, Journal of Biological Chemistry.

[14]  D. Prazeres,et al.  Translational Features of Human Alpha 2b Interferon Production in Escherichia coli , 2004, Applied and Environmental Microbiology.

[15]  P. Ho,et al.  Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli. , 2004, Protein expression and purification.

[16]  R. Kisilevsky,et al.  A Binding Site for Highly Sulfated Heparan Sulfate Is Identified in the N Terminus of the Circumsporozoite Protein , 2004, Journal of Biological Chemistry.

[17]  L. Young,et al.  Two-step total gene synthesis method. , 2004, Nucleic acids research.

[18]  A. Holder,et al.  Function of Region I and II Adhesive Motifs ofPlasmodium falciparum Circumsporozoite Protein in Sporozoite Motility and Infectivity* , 2002, The Journal of Biological Chemistry.

[19]  Ana Rodriguez,et al.  Invasion of mammalian host cells by Plasmodium sporozoites , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[20]  Y. Ikada,et al.  Liver targeting of interferon-beta with a liver-affinity polysaccharide based on metal coordination in mice. , 2001, The Journal of pharmacology and experimental therapeutics.

[21]  M. Bertini,et al.  Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor , 1997, Journal of viral hepatitis.

[22]  P. Vajro,et al.  Side effects of alpha-interferon therapy and impact on health-related quality of life in children with chronic viral hepatitis. , 1997, The Pediatric infectious disease journal.

[23]  T. J. Berkel,et al.  Targeting Hepatitis B Therapy to the Liver , 1996 .

[24]  T. V. van Berkel,et al.  Targeting hepatitis B therapy to the liver. Clinical pharmacokinetic considerations. , 1996, Clinical pharmacokinetics.