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

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

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 microg/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.

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Assessment of IL-10 integrity and dimerization. Viral (a) and murine (b) IL-10, purified from transgenic tobacco leaf extracts by IMAC, were analyzed by immunoblotting after separation by non-reducing (left panels) and reducing (right panels) SDS-PAGE. Commercial recombinant viral IL-10 produced in E. coli (rvIL-10) or murine IL-10 produced in insect cells (rmIL-10) were used as controls, and different amounts of plant-derived IL-10 were loaded, as indicated on top of each lane. (c) Elution profile of plant-derived mIL-10 from a Superdex 200 column showing a single peak which, compared to the elution volumes of standard reference molecules on the same columns (not shown), matches with the expected size of the dimer. Detection of IL-10 in the elution fractions was carried out by ELISA.
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Figure 3: Assessment of IL-10 integrity and dimerization. Viral (a) and murine (b) IL-10, purified from transgenic tobacco leaf extracts by IMAC, were analyzed by immunoblotting after separation by non-reducing (left panels) and reducing (right panels) SDS-PAGE. Commercial recombinant viral IL-10 produced in E. coli (rvIL-10) or murine IL-10 produced in insect cells (rmIL-10) were used as controls, and different amounts of plant-derived IL-10 were loaded, as indicated on top of each lane. (c) Elution profile of plant-derived mIL-10 from a Superdex 200 column showing a single peak which, compared to the elution volumes of standard reference molecules on the same columns (not shown), matches with the expected size of the dimer. Detection of IL-10 in the elution fractions was carried out by ELISA.

Mentions: The biological activity of IL-10 depends strictly on its dimeric state [19]. In order to determine whether or not mIL-10 and vIL-10 were properly assembled into dimers, the His6-tagged proteins were purified from stable transgenic tobacco leaves via immobilized-metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column, concentrated, and fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under either reducing or non-reducing conditions. Immunoblot analysis confirmed the correct molecular weights of both viral and murine IL-10 (19 and 21 kDa, respectively; Figures 3a and 3b, right panels). When separated under non-reducing conditions, the plant-derived vIL-10 predominantly formed stable dimers (Figure 3a, left panel), whereas mIL-10 was partly dimeric and partly monomeric (Figure 3b, left panel). Since commercial mIL-10 also gives rise to a monomer band under these conditions, this is most likely an artifact of the SDS-PAGE method. This was confirmed by gel filtration analysis, which showed that the plant-derived mIL-10 eluted as a single peak with a profile corresponding to the size of the dimeric form (Figure 3c).


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

Bortesi L, Rossato M, Schuster F, Raven N, Stadlmann J, Avesani L, Falorni A, Bazzoni F, Bock R, Schillberg S, Pezzotti M - BMC Biotechnol. (2009)

Assessment of IL-10 integrity and dimerization. Viral (a) and murine (b) IL-10, purified from transgenic tobacco leaf extracts by IMAC, were analyzed by immunoblotting after separation by non-reducing (left panels) and reducing (right panels) SDS-PAGE. Commercial recombinant viral IL-10 produced in E. coli (rvIL-10) or murine IL-10 produced in insect cells (rmIL-10) were used as controls, and different amounts of plant-derived IL-10 were loaded, as indicated on top of each lane. (c) Elution profile of plant-derived mIL-10 from a Superdex 200 column showing a single peak which, compared to the elution volumes of standard reference molecules on the same columns (not shown), matches with the expected size of the dimer. Detection of IL-10 in the elution fractions was carried out by ELISA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667500&req=5

Figure 3: Assessment of IL-10 integrity and dimerization. Viral (a) and murine (b) IL-10, purified from transgenic tobacco leaf extracts by IMAC, were analyzed by immunoblotting after separation by non-reducing (left panels) and reducing (right panels) SDS-PAGE. Commercial recombinant viral IL-10 produced in E. coli (rvIL-10) or murine IL-10 produced in insect cells (rmIL-10) were used as controls, and different amounts of plant-derived IL-10 were loaded, as indicated on top of each lane. (c) Elution profile of plant-derived mIL-10 from a Superdex 200 column showing a single peak which, compared to the elution volumes of standard reference molecules on the same columns (not shown), matches with the expected size of the dimer. Detection of IL-10 in the elution fractions was carried out by ELISA.
Mentions: The biological activity of IL-10 depends strictly on its dimeric state [19]. In order to determine whether or not mIL-10 and vIL-10 were properly assembled into dimers, the His6-tagged proteins were purified from stable transgenic tobacco leaves via immobilized-metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column, concentrated, and fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under either reducing or non-reducing conditions. Immunoblot analysis confirmed the correct molecular weights of both viral and murine IL-10 (19 and 21 kDa, respectively; Figures 3a and 3b, right panels). When separated under non-reducing conditions, the plant-derived vIL-10 predominantly formed stable dimers (Figure 3a, left panel), whereas mIL-10 was partly dimeric and partly monomeric (Figure 3b, left panel). Since commercial mIL-10 also gives rise to a monomer band under these conditions, this is most likely an artifact of the SDS-PAGE method. This was confirmed by gel filtration analysis, which showed that the plant-derived mIL-10 eluted as a single peak with a profile corresponding to the size of the dimeric form (Figure 3c).

Bottom Line: The best yields using this strategy in T1 plants were 10.8 and 37.0 microg/g fresh leaf weight for viral and murine IL-10, respectively.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scientific and Technologic Department, University of Verona, Strada Le Grazie 15, Verona, Italy. luisa.bortesi@univr.it

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 microg/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.

Show MeSH
Related in: MedlinePlus