Abstract
Porcine gamma interferon is a cytokine produced by activated T cells and NK cells with broad-spectrum antiviral activity and immunomodulatory function. However, pIFN-γ is a secretory protein that has a short half-life in organisms and is easily inactivated, making it difficult to apply widely in clinics. Therefore, we tried to optimize the expression of pIFN-γ in Pichia pastoris to obtain a large amount of highly active, easily purified pIFN-γ protein in vitro. Through C-terminal sequence analysis, we found a signal sequence (EKREAEAE) that was easily enzymolysed by a signal peptide enzyme, resulting in degradation and inactivation of the pIFN-γ protein. In this study, we optimized the pIFN-γ gene recombination sequence and mutated the 3' end of the pIFN-γ gene, resulting in a higher expression level and stronger biological activity, as well as a significant upregulation in the expression of the interferon-stimulated genes Mx1 and OAS1 in IPEC-J2 jejunal epithelial cells. Our data also showed that the fermentation process could significantly improve productivity. A recombinant Pichia pastoris strain with the optimized pIFN-γ gene could obtain a high yield of pIFN-γ protein, up to 9536 mg/L, after staged incubation for 0–24 h at 28°C, pH 6.0, and 50% dissolved oxygen (DO), followed by incubation for 24–72 h at 25°C, pH 6.0 and 30% DO. These data demonstrated, for the first time, that the expression level of pIFN-γ in Pichia pastoris was improved significantly by gene optimization with 3' end mutation and a fermentation process that maintained good biological activity, which is beneficial to the application of pIFN-γ in animal husbandry.
Highlights
Interferon gamma (IFN-γ) is a cytokine with antiviral and immunomodulatory functions produced by activated T cells and NK cells[1,2,3]
The Pichia pastoris expression system is an excellent eukaryotic expression system developed in recent years: it has a strong AOX promoter, an exogenous protein that has a certain function in translation modification, and the advantages of primitive nuclear biological molecular genetics and simple operation
There are some defects in the expression system of Pichia pastoris: some exogenous proteins are degraded by Pichia pastoris, there are still some exogenous proteins in the system that cannot be expressed or have low expression, and some exogenous proteins may have excessive glycosylation modification
Summary
Interferon gamma (IFN-γ) is a cytokine with antiviral and immunomodulatory functions produced by activated T cells and NK cells[1,2,3]. The total porcine interferon gamma IFN-γ (pIFN-γ) gene is 501 bases and encodes 166 amino acids. Enhance the production and biological activity of porcine interferon-γ the signal peptide, and 143 amino acids of pIFN-γ function in the activity of the mature protein[4]. The antiviral effect of interferon does not directly kill the virus but rather binds to cell surface receptors[6] to induce cells to produce enzyme-active antiviral proteins ADAR, PKR, Mx, OAS, and RNaseL, which degrade viral RNA, inhibit viral replication and translation, and inhibit viral shell formation to achieve antiviral effects[7,8,9]. The researchers used different expression systems to express pIFN-γ, but most of them have a problem with low protein expression or activity, which limits the further application of pIFN-γ[11]
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