Expression of Human Interferon-α8 Synthetic Gene under P_BAD Promoter

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Y. Mohammed¹, N. A. El-Baky¹, N. A. Redwan², and E. M. Redwan^1,2*

¹Antibody Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg El-Arab 21934, Alexandria, Egypt; fax: +203-459-3422; +203-459-3423; E-mail: redwan1961@yahoo.com

²Biological Science Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 23589, Saudi Arabia

^* To whom correspondence should be addressed.

Received February 14, 2012; Revision received August 4, 2012

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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 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.

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KEY WORDS: codon optimization, Escherichia coli, human interferon-alpha eight, synthetic gene

DOI: 10.1134/S0006297912100136

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