Transient expression of vacuolar antibodies and human transglutaminase in N. benthamiana leaves

OCAMPO, CAROLINA GABRIELA; MARIN VIEGAS, VANESA SOLEDAD; MAZZINI, FLAVIA; PETRUCCELLI, SILVANA*

Varadero

Congreso; Biotecnologia Habana; 2017

Centro de Ingeniería Genética y Biotecnología (CIGB)

Plant-based platforms are widely used for the expression of foreign proteins. To exploit its full potential, a better understanding of factors that influence the quantity and quality of foreign proteins is needed. In leaves, usually proteins are retained in the ER or sorted to the apoplast where an intense proteolysis occurs. Here we present the strategies develop to express antibodies (Ab) and human tissue transglutaminase (TG2). TG2, the main Celiac Disease (CD) autoantigen is a challenging protein since its toxicity. The first strategy tested was sorting to leaf central vacuoles. Both vac-Abs and vac-TG2 accumulated at levels 10-fold higher than their secretory counterparts. Although leaf central vacuole is considered a hostile environment intact vac-Abs and vac-TG2 were purified. Since N-glycan profile is very important for effectors Ab function, glycan analysis of vac-Ab and sec-Abs were performed. Vac-Ab´ s N-glycan profiling revealed the predominant presence of oligomannosidic (Man-7-9) next plant typical complex fucosylated and xylosylated GnGnXF structures, while sec-Ab carried mainly GnGnXF forms. The expected sub-cellular localization of sec-Abs and vac-Abs was confirmed by confocal microscopy. These data suggest that vac-Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab with Man-7-9) and trafficking through the Golgi (Ab with GnGnXF). A second strategy was the coexpression of a short elastin like polymer (ELP). A 2-fold increase on accumulation of ER and vac-TG2 was observed when ELP is coexpressed. In addition, if a single-chain-Ab fused to ELP is coexpressed a 5-fold increase on TG2 levels is achieved. Furthermore TG2 can be efficiently purified by inverse transition cycling. Plant purified TG2 was recognized by CD patients´IgA therefore is useful to develop CD screening assays. The combination of sorting strategies and coexpression of ELP results in an improvement of this leaf-based platform.