Planta

, Volume 231, Issue 4, pp 977–990 | Cite as

The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts

  • Inmaculada Farran
  • Iva McCarthy-Suárez
  • Francisco Río-Manterola
  • Cristina Mansilla
  • Juan José Lasarte
  • Ángel M. Mingo-Castel
Original Article

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.

Keywords

Extra domain A from fibronectin Molecular farming Plastid transformation Protein-fusion tag Vaccine adjuvant 

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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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Inmaculada Farran
    • 1
  • Iva McCarthy-Suárez
    • 1
  • Francisco Río-Manterola
    • 1
  • Cristina Mansilla
    • 2
  • Juan José Lasarte
    • 2
  • Ángel M. Mingo-Castel
  1. 1.Instituto de AgrobiotecnologíaUniversidad Pública de Navarra-CSIC-Gobierno de NavarraPamplonaSpain
  2. 2.Área de Terapia Génica y HepatologíaCIMA, Universidad de NavarraPamplonaSpain

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