title: Expression of human interferon-α8 synthetic gene under P(BAD) promoter.
abstract: Recombinant human interferon-α8 (rhIFN-α8) was obtained by synthesizing a codon-optimized gene in a two-step polymerase chain reaction (PCR) and expressing it in Escherichia coli. The gene encoding human IFN-α8 shows a high content of rare codons. These were replaced based on E. coli codon usage and balancing TA-GC ratio contents of the entire gene. The two-step PCR was performed using long (45-60 nucleotides) overlapped primers and two Taq polymerases (pfu clone and GC-rich system) and resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the IFN-α8 coding sequence; the pfu clone failed to amplify the gene in the correct size without unspecific bands. The full gene was cloned into the pBAD-TOPO expression vector. After cloning, the gene was reoriented by NcoI restriction digestion and religation. The ligated pBAD-TOPO-IFN-α8 (pBAD-IFNα8) plasmid carried the IFN-α8 gene under transcriptional control of the L-arabinose-inducible P(BAD) promoter. IFN-α8 expression was optimized with respect to L-arabinose concentration, temperature, and time of induction in shake flask cultures to maximize the yield of soluble IFN-α8. The produced IFN-α8 was characterized by polyacrylamide gel electrophoresis and immunoassays. After purification on DEAE-Sepharose, the yield was 100 mg/liter. The antiviral and anticancer activities of the IFN-α8 were evaluated in comparison with IFN-α2a, and the results are discussed.
authors.affiliation: Antibody Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Alexandria, Egypt.
authors.forename: Y
authors.initials: Y
authors.lastname: Mohammed
authors.forename: N A
authors.initials: NA
authors.lastname: El-Baky
authors.forename: N A
authors.initials: NA
authors.lastname: Redwan
authors.forename: E M
authors.initials: EM
authors.lastname: Redwan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D005813:Genes, Synthetic / Q000235:genetics*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000187:drug effects; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D000998:Antiviral Agents; D017405:Herpes Simplex Virus Protein Vmw65; C554164:IFNA8 protein, human; D016898:Interferon-alpha; D011994:Recombinant Proteins