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.
Highlights
Interleukin 6 (IL6) is a cytokine with several important physiological roles, including the regulation of immune responses, inflammation and hematopoiesis [1]
Clinical studies have demonstrated that IL6 is a useful therapeutic target for certain immune-mediated inflammatory diseases (IMIDs) [8], but the development of novel drugs has been delayed by the limited availability of recombinant IL6
We found that the MagnICON transient expression system produced much higher yields of IL6 than transgenic plants and that N. benthamiana was the most suitable host, achieving up to 7% TSP compared to 1% in cv
Summary
Interleukin 6 (IL6) is a cytokine with several important physiological roles, including the regulation of immune responses, inflammation and hematopoiesis [1]. Excess IL6 is associated with various immune-mediated inflammatory diseases (IMIDs), including rheumatoid arthritis [2], atherosclerosis [3] and the neurodegenerative cascade leading to Alzheimer’s disease [4]. Clinical studies have demonstrated that IL6 is a useful therapeutic target for certain IMIDs [8], but the development of novel drugs has been delayed by the limited availability of recombinant IL6. Therapeutic proteins are generally manufactured in Escherichia coli, yeast or mammalian cells, despite the limited scalability of these platforms [9,10]. Complex proteins produced in E. coli tend to accumulate within inclusion bodies and these require laborintensive resolubilization procedures that are generally avoided in commercial downstream processing [11,12]. An alternative eukaryotic expression platform is required to provide sufficient amounts of soluble and biologically-active IL6
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