Expression of human interferon-α8 synthetic gene under PBAD promoter
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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 PBAD 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.
Key wordscodon optimization Escherichia coli human interferon-alpha eight synthetic gene
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