Production of Human IFNγ Protein in Nicotiana benthamiana Plant through an Enhanced Expression System Based on Bamboo mosaic Virus.

Min-Chao Jiang,Chung-Chi Hu,Na-Sheng Lin,Yau-Heiu Hsu

doi:10.3390/v11060509

Abstract

Plant-based systems are safe alternatives to the current platforms for the production of biologically active therapeutic proteins. However, plant-based expression systems face certain major challenges, including the relatively low productivity and the generation of target proteins in biologically active forms. The use of plant virus-based expression systems has been shown to enhance yields, but further improvement is still required to lower the production cost. In this study, various strategies were employed to increase the yields of an important therapeutic protein, human interferon gamma (IFNγ), in Nicotiana benthamiana through modifications of expression vectors based on potexviruses. Among these, the vector based on a coat protein (CP)-deficient Bamboo mosaic virus (BaMV), pKB△CHis, was shown to exhibit the highest expression level for the unmodified IFNγ. Truncation of the N-terminal signal peptide of IFN (designated mIFNγ) resulted in a nearly seven-fold increase in yield. Co-expression of a silencing suppressor protein by replacing the coding sequence of BaMV movement protein with that of P19 led to a 40% increase in mIFNγ accumulation. The fusion of endoplasmic reticulum (ER) retention signal with mIFNγ significantly enhanced the accumulation ratio of biologically active dimeric mIFNγ to 87% relative to the non-active monomeric form. The construct pKB19mIFNγER, employing the combination of all the above enhancement strategies, gave the highest level of protein accumulation, up to 119 ± 0.8 μg/g fresh weight, accounting for 2.5% of total soluble protein (TSP) content. These findings advocate the application of the modified BaMV-based vector as a platform for high-level expression of therapeutic protein in N. benthamiana.

Highlights

IntroductionPlant molecular farming (PMF) is a cost-effective technology that uses plants to produce valuable pharmaceutical agents, such as therapeutic proteins, antibodies, enzymes, and edible vaccines [1,2,3]
Plant molecular farming (PMF) is a cost-effective technology that uses plants to produce valuable pharmaceutical agents, such as therapeutic proteins, antibodies, enzymes, and edible vaccines [1,2,3].Currently the plant-based systems are emerging as an alternative bio-production system to microbial and animal expression systems for the advantages in many aspects [3,4,5,6]
It has been previously shown that nucleotides +1 to +15 at the 50 -terminal of Bamboo mosaic virus (BaMV)-S strain coat protein (CP) open reading frame (ORF) are essential for CP subgenomic RNA promoter activity (SGP) [35]

Summary

Introduction

Plant molecular farming (PMF) is a cost-effective technology that uses plants to produce valuable pharmaceutical agents, such as therapeutic proteins, antibodies, enzymes, and edible vaccines [1,2,3]. The plant-based systems are emerging as an alternative bio-production system to microbial and animal expression systems for the advantages in many aspects [3,4,5,6]. Plant-based production systems offer the potential for low risk of animal pathogen contamination, eukaryotic post-translational modification, direct oral consumption of the expressed protein, low cost of biomass production, and rapid scalability [3]. The low yields in some plant production systems have posed a significant challenge to the application of PMF-based pharmaceuticals at an industrial scale [8,9,10].

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