High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

Abstract

Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T0 transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T2 plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein.

High-level expression of sucrose inducible sweet potato sporamin gene promoter: &#x3b2;-glucuronidase fusion gene in transgenic <i>Nicotiana plumbaginifolia</i>

“…Therefore, we have confirmed that Spo min is an excellent promoter for making plant bioreactors. Previous reports have shown that ER is a suitable location to accumulate heterologous protein in transgenic Nicotiana plants leaves (Benchabane et al 2008;Hamorsky et al 2013;Nausch et al 2012;Yang et al 2005). Our study also showed that ER was suitable for accumulating heterologous GUS protein in an expression system using Spo min and N. plumbaginifolia and also that leaves might be a more suitable organ than stems and roots because of high GUS activities of SE, especially in SE leaves (Figure 3).…”

“…These results are supported by previous reports. For example, Nausch et al (2012) showed that accumulation of human interleukin 6 was over 10 times greater in ER than in apoplasts or vacuoles, comprising up to 0.005% of TSP in N. benthamiana leaves. Also, in maize seeds, the accumulation of heat-labile toxin was about 5 times higher in ER than in plastids, reaching 0.0028% of TSP (Streatfield et al 2003).…”

The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms

“…1D). However, PVX failed to elicit necrosis in the presence of HCwt, likely due to the fact that transient expression of PVX is less efficient in tobacco than in N. benthamiana (44).…”

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

“…Using the same sorting strategy, human interleukin (IL) 6 was expressed in stable transgenic N. tabacum and temporally in Nicotiana benthamiana, but ER sorted-IL6 produced yields 6.25 higher than vac-IL6, although IL6 was also fused to AFVY Ct VSS. 29 ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP and none cytotoxic effect were observed. 29 Deposition of antibodies (Abs) in vacuoles of vegetative tissues had also been studied and information about trafficking and modifications in different compartments was obtained based on its N-glycosylation pattern.…”

“…29 ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP and none cytotoxic effect were observed. 29 Deposition of antibodies (Abs) in vacuoles of vegetative tissues had also been studied and information about trafficking and modifications in different compartments was obtained based on its N-glycosylation pattern. The N-glycosylation of proteins starts in the ER with the transfer of the Glc3Man9GlcNAc2 oligosaccharide to a specific Asn residues on the nascent polypeptide followed of a limited trimming in both the ER and Golgi and sequential addition of monosaccharides, as the protein travel through the Golgi complex, to yield complex N-glycans, typically GlcNAc2Man3FucXylGlcNAc2 structures (Fig.…”