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.

Min-Chao Jiang,Yau-Heiu Hsu,Wei-Li Hsu,Chung-Chi Hu,Tsui-Ling Hsu,Na-Sheng Lin

doi:10.3389/fpls.2020.594758

Abstract

Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.

Highlights

Plants have been developed recently as bioreactors for the industrial-scale production of therapeutic proteins, such as Elelyso R (β-glucocerebrosidase) (Zimran et al, 2015), ZMappTM (Ebola monoclonal antibodies) (Olinger et al, 2012; Chen and Davis, 2016)
To address the low yield issue, we have previously developed a Bamboo mosaic virus (BaMV)-based overexpression vector with the co-expression of a silencing suppressor P19, designated pKB19, which was shown to significantly increase the yield of a vaccine candidate in transgenic Nicotiana benthamiana suspension cell cultures (Muthamilselvan et al, 2016) or various forms of recombinant human interferon gamma (IFNγ) proteins transiently expressed in inoculated N. benthamiana plants (Jiang et al, 2019)
SSPr1 is derived from N. benthamiana, it has not been assayed for the function in enhancing the solubility or secretion efficiency of target proteins (TPs)

Summary

Introduction

Plants have been developed recently as bioreactors for the industrial-scale production of therapeutic proteins, such as Elelyso R (β-glucocerebrosidase) (Zimran et al, 2015), ZMappTM (Ebola monoclonal antibodies) (Olinger et al, 2012; Chen and Davis, 2016). The relatively low yields and the difficulties in downstream processing of PMPs remain major challenges which limit the industrial or commercial acceptance of plant production systems (Schillberg et al, 2019). To address the low yield issue, we have previously developed a Bamboo mosaic virus (BaMV)-based overexpression vector with the co-expression of a silencing suppressor P19, designated pKB19, which was shown to significantly increase the yield of a vaccine candidate in transgenic Nicotiana benthamiana suspension cell cultures (Muthamilselvan et al, 2016) or various forms of recombinant human interferon gamma (IFNγ) proteins transiently expressed in inoculated N. benthamiana plants (Jiang et al, 2019). Various strategies applied were effective in increasing the yields of dimeric (D) forms of recombinant interferon gamma IFNγ proteins, such as mature IFNγ (mIFNγ) and mIFNγER (mIFNγ fused with ER retention signal) (Jiang et al, 2019), the lower

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