A reporter system to study the capacity of plant cell to produce full length immunoglobulins in the secretory pathway

SILVINA MANGANO, CINTIA DANIELA GONZÁLEZ AND SILVANA PETRUCCELLI*.

Havana

Congreso; International Congress Biotechnology Havana 2011; 2011

Abstract: Most of the biotechnological important proteins are synthesized on the secretory pathway (SP); therefore increasing the cell´s capacity to properly fold and transport proteins is of critical importance in developing of expression platforms systems. Molecular chaperones and foldases have a tissue specific expression patterns what suggest that the molecular machine that facilitate folding and assembly is protein specific. The aim of this work was to develop reporter systems to study factors that can modify the capability of plant cells to synthesize proteins on the SP. To this end, the light and heavy chains (LC and HC) of the catalytic antibody 14D9 were fused to different fluorescent proteins and the constructs were studied by transient expression in Nicotiana benthamiana. As a control a secretory version of the fluorescent protein was also produced. In temporal expression assay, abaxial epidermal leaf cells were observed by confocal scanning microscopy. The HC-eYFP expressed alone had a reticular pattern and LC-mCherry expressed alone was found mainly outside the cells and a minor proportion in central vacuole. When the LC-mCherry and HC-eYFP were expressed together, red fluorescence was found mainly in vacuoles and not on the limits of cells. The modification of LC-mCherry localization is an indication of interaction between HC and LC. This interaction and activity was confirmed by Western Blot analysis and Elisa assay, respectively. Since, assembly and transport through the SP are required for the mAb-FP to reach the vacuole, these constructs will be useful as a reporter system to study genes that could increase immunoglobulin accumulation. To test these reporter systems, plant cells were subject to stress an impact on the accumulation levels of the mAb-FP and secFP were analyzed corroborating the ability of the systems to response to factors that can increase plant cell folding capacity. The developed systems can be used to identify key regulatory gene of the SP