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-α (TNF-α) 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.
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Abbreviations
- BMDC:
-
Bone marrow-derived dendritic cell
- DC:
-
Dendritic cell
- DS:
-
Downstream sequence
- EDA:
-
Extra domain A from fibronectin
- TCP:
-
Total cellular protein
- TLR:
-
Toll-like receptor
- TNF-α:
-
Tumour necrosis factor-α
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Acknowledgments
The authors wish to thank P. Obregon and J. Veramendi, UPNA, Pamplona, Spain for a critical reading of this manuscript. The authors also thank K. Recalde and I. Serrano, UPNA, Pamplona, Spain for technical assistance. This work was supported by a grant (Proyecto EUROINNOVA) from the Department of Innovation (Gobierno de Navarra).
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I. Farran and I. McCarthy-Suárez contributed equally to this article.
The authors dedicate this publication to the memory of their colleague and co-author, Dr. Ángel M. Mingo-Castel, who died on 7 June 2009.
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Farran, I., McCarthy-Suárez, I., Río-Manterola, F. et al. The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts. Planta 231, 977–990 (2010). https://doi.org/10.1007/s00425-010-1102-4
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DOI: https://doi.org/10.1007/s00425-010-1102-4