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

Abstract

Most of the biotechnological important proteins are synthesized on the secretory pathway; 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, and in particular full length antibodies, on the secretory pathway. 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 both transient and stable expression in Nicotiana benthamiana and Arabidopsis thaliana. 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 secretory pathway 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 (temperature and UV light) 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 secretory pathway