Synonymous Codon Usage Bias And Overexpression Of A Synthetic Gene Encoding Interferon α2b In Pichia Pastoris

The interferons are a group of natural antiviral substances discovered in 1957. Differential activities of IFN subtypes have been reported. Especially, the human interferonα2b, the most widely used member of IFN-αfamily, influence many biological processes including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. This has led to the use of IFN-α2b as potential agents for therapy of neoplastic diseases, cancers and hepatitis.Pichia expression system (Pichia pastoris/pPIZα) is widely used in recombinant protein expression. The system can be used in a continuous, high density fermentation induced by methanol. Although the final yield of a protein is greatly influenced by its inherent properties, the yield could be significantly enhanced by manipulation of the factors that influence gene expression and production stability. To increase IFN-α2b of industrial yields, the optimized strategies for enhancing the production level of proteins and synonymous codon usage bias in the P. pastoris expression system were applied in this study. Some former study demonstrated that the use of P. pastoris synonymous codon usage bias in some different kind of gene could increase production and activity of the proteins correspondingly.In this study, to achieve higher level of Interferonα2b (IFN-α2b) expression in the methylotrophic yeast Pichia pastoris, the cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P.pastoris and the optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into Pichia pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allows the expression of the IFN-α2b transformant up to 810 mg/L detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×105 IU/mL.