AUTHOR=Jiang Min-Chao , Hsu Wei-Li , Tseng Ching-Yu , Lin Na-Sheng , Hsu Yau-Heiu , Hu Chung-Chi 

TITLE=Development of a tag-free plant-made interferon gamma production system with improved therapeutic efficacy against viruses

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1341340

DOI=10.3389/fbioe.2023.1341340

ISSN=2296-4185

ABSTRACT=<p>Plants offer a promising platform for cost-effective production of biologically active therapeutic glycoproteins. In previous studies, we have developed a plant expression system based on <italic>Bamboo mosaic virus</italic> (BaMV) by incorporating secretory signals and an affinity tag, which resulted in notably enhanced yields of soluble and secreted fusion glycoproteins (FGs) in <italic>Nicotiana benthamiana</italic>. However, the presence of fusion tags on recombinant glycoproteins is undesirable for biomedical applications. This study aimed to develop a refined expression system that can efficiently produce tag-free glycoproteins in plants, with enhanced efficacy of mature interferon gamma (mIFNγ) against viruses. To accommodate the specific requirement of different target proteins, three enzymatically or chemically cleavable linkers were provided in this renovated BaMV-based expression system. We demonstrated that <italic>Tobacco etch virus</italic> (TEV) protease could process the specific cleavage site (L<sub>TEV</sub>) of the fusion protein, designated as SS<sup>Ext</sup>His(SP)<sub>10</sub>L<sub>TEV</sub>-mIFNγ, with optimal efficiency under biocompatible conditions to generate tag-free mIFNγ glycoproteins. The TEV protease and secretory-affinity tag could be effectively removed from the target mIFNγ glycoproteins through Ni<sup>2+</sup>-NTA chromatography. In addition, the result of an antiviral assay showed that the tag-free mIFNγ glycoproteins exhibited enhanced biological properties against <italic>Sindbis viru</italic>s, with comparable antiviral activity of the commercialized HEK293-expressed hIFNγ. Thus, the improved BaMV-based expression system developed in this study may provide an alternative strategy for producing tag-free therapeutic glycoproteins intended for biomedical applications.</p>