congresos y reuniones científicas
Construction of a reporter system to study factors affecting the synthesis of full lenght inmunoglobulins in plant cells
Congreso; Fourth International Conference- Plant Based Vaccines and Antibodies- PBVA 2011; 2011
Production of heterologous proteins in plants and other eukaryotic hosts is often restricted by inefficient folding and secretion of the product. To overcome this limitation, a frequently successful strategy exploited in yeast and mammalian cell lines, consists in the overexpression of proteins implicated in folding and/or transport through the secretory pathway. Since broad functional categories of genes, such as chaperones, cytoskeletal, cell signaling, metabolic, and mitochondrial are involved in the processes of protein synthesis, folding and secretion, the development of reporter systems is of interest. These systems will facilitate the identification of genes controlling these processes. Taking into account that some proteins implicated in folding are specific, the aim of this work was to develop a reporter system to study factors that can modify the capability of plant cells to synthesize full length antibodies. To this end, the light and heavy chains (LC and HC) of the catalytic antibody 14D9 were fused to fluorescent proteins (mCherry transient and stable expression in Nicotiana benthamiana and Arabidopsis thaliana. In temporal expression assays, abaxial epidermal leaf cells were observed three days after agroinfiltration by confocal scanning microscopy. The HC-eYFP expressed alone had a reticular pattern and colocalized with RFP-HDEL suggesting that HC alone can not travel further of the endoplasmic reticulum (ER). In contrast, LC-mCherry expressed alone was found mainly outside the cells and a minor proportion in central vacuole. Plasmolysis of the plant cells by treatment with mannitol confirmed that LC-mCherry accumulated in the apoplastic space. Unlike LC-mCherry, LC-tDtomato was found in the ER, therefore the fusion of LC to a dimeric fluorescent protein alters protein sorting. When the LC-mCherry and HC-eYFP were expressed together, red fluorescence was found mainly in vacuoles. Not red fluorescence was observed on the limits of cells expressing simultaneously both LC-mCherry and HC-eYFP, the modification of LC-mCherry localization is an indication of interaction between HC and LC. This interaction was confirmed by Western Blot analysis. Temporal expression of LC-mCherry in transgenic HC-eYFP arabidopsis plants also gave red fluorescence mainly in vacuoles. Although, secretion of the full length immunoglobulin fused to mCherry and eYFP was expected, the mAb-FP was found in central vacuole. Since, assembly and transport through the secretory pathway are required for the mAb-FP to reach the vacuole, transgenic Arabidopsis plants expressing these constructs will be useful as a reporter system to screen, using different genomic approaches, for genes that could increase immunoglobulin accumulation