Subcellular targeting and induction of protein bodies as strategies to increase human transglutaminase´s yields in Nicotiana benthamiana leaves

MARIN VIEGAS, VANESA SOLEDAD; ACEVEDO, GONZALO RAÚL; CHIRDO, FERNANDO GABRIEL; PETRUCCELLI, SILVANA

Foz Iguazu

Congreso; International Plant Molecular Biology Congress (IPMB); 2015

International Society for Plant Molecular Biology (ISPMB)

Human tissue transglutaminase (TG2) is the main Celiac Disease (CD) autoantigen and presence of IgA antibodies against TG2 in sera is an important diagnostic marker. CD is considerably underdiagnosed and availability of economic TG2-Ig A tests is needed. Since TG2 is a hard to produce protein in different expression systems we aimed to develop plant-based transient expression systems to produce TG2.In this work, a transient expression system for the production of TG2 in Nicotiana benthamiana leaves was optimized. 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 is obtained when vac-TG2 and ER-TG2 constructs are 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 TG2 specific monoclonal antibodies (mAbs) that recognize different epitopes and sera of CD patients, proving that plant produced antigen has similar immunochemical characteristic than human TG2. 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. Notably, the combination of p19 post-transcriptional gene silencing suppressor and a protein body inducing construct increased recombinant protein yields. Moreover the two plants purified TG2 versions are recognized by TG2-specific mAbs and sera of CD patients. This strategy could be extended to other problematic proteins enforced the advantages of plant based production platforms.