Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?

Abstract

Human interferon gamma (hIFNγ) is an important cytokine in the innate and adaptive immune system, produced commercially in Escherichia coli. Efficient expression of hIFNγ has been reported once for Pichia pastoris (Wang et al., 2014) - a proven heterologous expression system. This study investigated hIFNγ expression in P. pastoris replicating the previous study and expanding by using four different strains (X33: wild type; GS115: HISMut; KM71H: Arg, Mut and CBS7435: Mut) and three different vectors (pPICZαA, pPIC9 and pPpT4αS). In addition, the native sequence (NS) and two codon-optimised sequences (COS1 and COS2) for P. pastoris were used. Methanol induction yielded no expression/secretion of hIFNγ in X33, highest levels were recorded for CBS7435: Mut (∼16 μg. L). mRNA copy number calculations acquired from RT-qPCR for GS115-pPIC9-COS1 proved low abundance of mRNA. A 10-fold increase in expression of hIFNγ was achieved by lowering the minimal free energy of the mRNA and 100-fold by Mut phenotypes, substantially lower than reported by Wang et al. (2014). We conclude that commercial production of low cost, eukaryotic recombinant hIFNγ is not an economically viable in P. pastoris. Further research is required to unravel the cause of low expression in P. pastoris to achieve economic viability.

Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain

“…Here we have demonstrated an approach whereby easily acquired genomic and transcriptomic data on variants of a given organism enable prediction of performance that can guide selection of an optimal host. To date, the varied relative performance of P. pastoris strains has been reported with respect to product quality, titer, and growth (Blanchard et al, ; Huang et al, ; Razaghi et al, ). Our analyses revealed the integrity of the cell wall likely confers the largest source of variation in performance among these strains.…”

Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System

“…The N-glycosylation modification is also critical for the half-life of mIFNγ in the bloodstream, affecting its therapeutic usefulness (Hooker and James, 1998;Razaghi et al, 2016). It has been shown that the unglycosylated form of mIFNγ monomers are prone to aggregation through the interactions of the hydrophobic domains, leading to lower solubility (Razaghi et al, 2016) and adding more difficulties for downstream purification processes, such as denaturing and refolding (Jin et al, 2006;Petrov et al, 2010;Razaghi et al, 2017a). Thus, further improvement of the BaMV-based expression system is required for industrial applications.…”

IFN-γ establishes interferon-stimulated gene-mediated antiviral state against Newcastle disease virus in chicken fibroblasts

“…GO and KEGG pathway enrichment analyses revealed distinct biological features in all treatment groups. In order to replicate in the host cells, viruses need to breach the immune and metabolic processes of the host cells [38]. We found this to be the case when we observed significant variations in cellular metabolic processes (i.e.…”

“…Standardization of expression conditions and a lack of glycosylation render these IFN-γ preparations more protease-resistant compared with the glycosylated forms that have a shorter half-life in the blood. Endotoxin contamination, inclusion body formation, and complex protein purification from E. coli increase production costs of E. coli-derived IFN-γ [37,38].…”