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Recombinant human interferon α-2b is an FDA-approved drug for monotherapy or in combination therapy with other drugs for hepatitis and cancers. It belongs to a family of homologous proteins involved in antiviral, antiproliferative, and immunoregulatory processes. A different expression system has been used for overexpression of this protein. Escherichia coli expression system is a highly characterized host and various expression settings have been developed based on its properties. However, finding the best conditions for the overexpression of recombinant human interferon α-2b remains to be addressed. In this study, the expression of synthetic human interferon α-2b gene in Escherichia coli was greatly improved by adjusting the expression condition. In this regard, a recombinant gene was designed and codon optimized for the periplasmic expression of this protein. Then, gene subcloning was employed to insert the synthesized gene into the pET22b expression vector. Thereafter, the response surface methodology was employed to design 20 experiments to find out the optimum points for isopropyl β-D-1-thiogalactopyranoside concentration, post-induction period, and the cell density of induction (OD600). The expression fluctuations were assessed by using the real-time polymerase chain reaction method. Our results indicated that the synthetic human interferon α-2b gene was successfully codon optimized and subcloned into the expression vector. The realtime polymerase chain reaction results revealed that the optimum levels of the selected parameters are 0.27 mM for isopropyl β-D-1-thiogalactopyranoside concentration, 7.98 H for the post-induction period, and 3.93 for cell density (OD600). These optimized conditions led to a 3.5-fold increase in the rhIFNα2b expression, which is highly promising for large scale rhIFNα2b overexpression. [ABSTRACT FROM AUTHOR]

Recombinant human interferon-β (rhIFN-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often laborintensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-β analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-β analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-β (Opt-IFN-β) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-β, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-β had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-β. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-β analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy. [ABSTRACT FROM AUTHOR]