ABSTRACT
We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0.3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-alpha (TNF-alpha) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant.
Subject(s)
Adjuvants, Anesthesia/pharmacology , Chloroplasts/metabolism , Fibronectins/metabolism , Fibronectins/pharmacology , Nicotiana/metabolism , Plants, Genetically Modified/metabolism , Adjuvants, Anesthesia/metabolism , Animals , Blotting, Northern , Blotting, Southern , Cells, Cultured , Chloroplasts/genetics , Chromatography, Ion Exchange , Dendritic Cells/drug effects , Fibronectins/genetics , Mice , Plants, Genetically Modified/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Nicotiana/geneticsABSTRACT
Histidine-tagged human cardiotrophin-1 (hCT-1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5'-untranslated regions (5'-UTRs) (psbA and phage T7 gene 10). The psbA 5'-UTR promotes recombinant hCT-1 (rhCT-1) accumulation in chloroplasts at higher levels (eight-fold) than those obtained for the phage T7 gene 10 5'-UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT-1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5'-UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (approximately 35%) than that of commercial hCT-1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT-1, suggesting that 32 h of over-lighting affects the biological activity of rhCT-1. Because high levels of rhCT-1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a 'closed system' unit, yielding up to 3.2 kg per year of rhCT-1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT-1 for liver transplantation surgery in a cost-effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant-based biopharmaceuticals.
