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

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.

Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking system

“…Previous studies utilized multiple plant species, tissue types, and expression systems for recombinant protein production 40,41 . Majority of them used transient Agrobacterium-and viral vector-based approaches in Nicotiana benthamiana or N. tabacum [42][43][44][45][46] . While the transient systems are viable approaches, they are technically feasible only in few plant species that are amenable for infiltration and/or are hosts for the viruses used as viral vectors.…”

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 bottom panel shows the amount of rRNA in each sample, stained with Ethidium bromide (EtBr) as the loading control. dilution) were used for the determination of TP expression levels of different BaMV-based vectors as described previously (Jiang et al, 2019).…”

“…The epitopepresenting chimeric BaMV virions were not designed to be secreted to the medium of the suspension cell cultures (Muthamilselvan et al, 2016), and had to be purified from the cultured biomass. On the other hand, although 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 Abbreviations: AWF, apoplast washing fluid; BaMV, Bamboo mosaic virus; CaMV, Cauliflower mosaic virus; CBS, Coomassie Blue stain; cGMP, current Good Manufacturing Practice; CP, coat protein; D, dimeric; DG, dimeric glycosylated mIFNγ; DPI, days post-infiltration; DSP, during downstream purification; FW, fresh weight; Glyco-mIFNγ, glycosylated mIFNγ; HypGPs, hydroxyproline (Hyp)-O-glycosylated peptides; IAV, Influenza A virus; IB, immunoblotting; IC, intracellular; IFNγ, interferon gamma; LC-MS/MS, Liquid chromatographytandem mass spectrometry; Mγ, monomeric mIFNγ; M, monomeric; MG, monomeric glycosylated mIFNγ; mIFNγ, mature interferon gamma; M r , molecular weights; P1, pellet fraction after centrifugation at 1,000 × g; P30, pellet fraction after centrifugation at 30,000 × g; PAS, periodic acid-Schiff stain; PMPs, plant made-pharmaceuticals; PNGase A, Peptide-N-Glycosidase A; PNGase F, Peptide-N-Glycosidase F; PTM, post-translational modification; RuBisCO, Ribulose-1,5-bisphosphate carboxylase/oxygenase; S1, soluble fraction after centrifugation at 1,000 × g; S30, soluble fraction after centrifugation at 30,000 × g; SINV, Sindbis virus; SS(s), secretory signal(s); TP(s), target protein(s); TSP, total soluble protein.…”

“…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 ). However, the downstream processing of the target proteins (TPs) is still challenging.…”

“…The epitope-presenting chimeric BaMV virions were not designed to be secreted to the medium of the suspension cell cultures ( Muthamilselvan et al, 2016 ), and had to be purified from the cultured biomass. On the other hand, although 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 solubility and lesser N-glycosylation level of the TPs limited their commercial applications as pharmaceuticals. The native human IFNγ is a well-characterized secretory, dimeric glycoprotein (D glycoprotein) generated by the dimerization of two IFNγ monomers with 0, 1, or 2 N-linked glycan each at the two potential glycosylation sites, residues N 25 and N 97 ( Sareneva et al, 1994 , 1995 ).…”