Research article

Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco

Luisa Bortesi^1,8 , Marzia Rossato² , Flora Schuster³ , Nicole Raven⁴ , Johannes Stadlmann⁵ , Linda Avesani^1,8 , Alberto Falorni⁶ , Flavia Bazzoni² , Ralph Bock⁷ , Stefan Schillberg⁴ and Mario Pezzotti^1,8

¹Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

²Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy

³Institute for Molecular Biotechnology, Biology VII, RWTH, Worringerweg 1, 52074 Aachen, Germany

⁴Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany

⁵Department for Chemistry, Glycobiology Division, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria

⁶Department of Internal Medicine, University of Perugia, Via E. Dal Pozzo, 06126 Perugia, Italy

⁷Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany

⁸Department for Sciences, Technologies and Markets of Grapevine and Wine, University of Verona, Via della Pieve 70, 37029 San Floriano di Valpolicella (VR), Italy

author email corresponding author email

BMC Biotechnology 2009, 9:22doi:10.1186/1472-6750-9-22

Published:	19 March 2009

Abstract

Background

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results

Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T₁ plants were 10.8 and 37.0 μg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion

Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.