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The interferons are a group of natural antiviral substances discovered in 1957 (7). Differential activeties of IFN subtypes have been reported (2). In particular, human interferon α2b, the most widely used member of IFN-α family, influences many biological processes including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. The curative effect has led to the use of IFN-α2b as potential agents for therapy of neoplastic diseases, cancers and hepatitis (6, 8, 10, 15).
The powerful Pichia pastoris/pPICZα eukaryotic expression system, which can grow rapidly at high densities, was used to express the recombinant proteins at high-levels. The expression system carries the strong alcohol oxidase Ⅰ (AOX1) promoter, the mating factor signal sequence from S. cerevisiae and the Zeocin selectable marker (14). Although the final yield of a protein is greatly influenced by its inherent properties, the yield can be significantly enhanced by manipulation of the factors that influence gene expression and production stability (1).
To increase the expression level of IFN-α2b, 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. Many studies have demonstrated that the use of P. pastoris synonymous codon usage bias in a variety of genes could result in high expression level and enhanced activity of the relevant gene products (12, 13, 16).
Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast
- Received Date: 22 November 2006
- Accepted Date: 17 January 2007
Abstract: To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. 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 allowed expression of the IFN-α2b transformant up to 810 mg/L as 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.