Production of the main celiac disease autoantigen by transient expression in Nicotiana benthamiana.

VANESA SOLEDAD MARIN VIEGAS; GONZALO RAÚL ACEVEDO; MARIELA PAULA BAYARDO; CHIRDO FERNANDO GABRIEL; PETRUCCELLI SILVANA

Frontiers in Plant Science

Frontiers

Año: 2015 p. 1 - 11

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen: human tissue transglutaminase (TG2), is a challenger for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein.In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and performance of plant produced TG2 in CD screening test was evaluated. To reduce TG2 toxic effects a subcellular targeting strategy was tested. Endoplasmic reticulum (ER) retained TG2 and a vacuolar (vac) sorted TG2 accumulated at higher levels than their cytosolic and secretory counterparts. The highest yield was obtained when vac-TG2 and ER-TG2 constructs were coinfiltrated with a protein body (PB) inducing construct based in an elastin-like polymer and the post transcriptional suppressors of gene silencing p19. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bound different epitopes proving that plant produced antigen has similar immunochemical characteristics than human TG2. Finally, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients, confirming the usefulness of these plant-produced TG2 versions.In conclusion, this work shows that both vacuolar and ER versions of this large, toxic and autoproteolytic protein can be easily produced and purified from the leaves. In addition, that the combination of p19 post transcriptional gene silencing suppressor and a protein body inducing construct increased recombinant protein yields. Importantly the two plant- purified TG2 versions are recognized by TG2-specific mAbs and serum IgA from CD patients while are not recognized by serum from non-celiac controls. These results point out that plant produced antigens are useful to develop screening assays. This strategy could be extended to other problematic proteins enforced the advantages of plant based production platforms.