Abstract
Recombinant human consensus interferon-alpha (cIFN-alpha) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-alpha gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45-50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-alpha gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-alpha (Power3-cIFN alpha) plasmid carried the cIFN-alpha gene under transcriptional regulation of the heat-inducible lambda P-L promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-alpha protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-alpha protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-alpha anti-cancer activity was assayed and compared with the commercially available IFN-alpha 2a.