(164j) Engineer Novel Functional Proteins in Plant Cell Culture for Industrial and Biomedical Applications

Genetic engineering of plants and plant cells has been used for generating transgenic plants with new traits or for “molecular farming” to produce valuable recombinant proteins. We aim to leverage two unique post-translational modifications – “glycosylphosphatidylinositol (GPI) anchor” and “hydroxyproline (Hyp)-O-glycosylation” – to strategically design and engineer functional recombinant proteins in plants and plant cells for industrial and biomedical applications. GPI anchoring leads to the attachment of glycolipid anchor to the C-terminus of a protein, which was found to dramatically promote the intracellular transport of anchored proteins in plant cells and display the proteins on the cell surface. GPI anchoring modification also facilitated N-linked and Hyp-O-linked glycosylation, resulting in more homogeneous glycosylation of the anchored proteins. Hyp-O-glycosylation is a plant-specific posttranslational modification that involves the addition of the arabino-oligosaccharides or arabinogalactan polysaccharides to the residues. Hyp-O-glycosylation was found to facilitate extracellular secretion of associated proteins and protect proteins from proteolytic degradation. For an industrial application, the GPI anchor and a Hyp-O-glycosylation module (SP)₂₀ consisting of 20 repeats of the “Ser-Pro” motif was engineered to the thermophilic E1 endoglucanase (cellulase) in tobacco plants to reconstruct the plant cell wall for enhanced processability of the cell wall biomass, leading to more than 50% increase of the saccharification efficiency. The two functional modules were also engineered to an anti-TNFα molecule (single-chain fragment variable of the human anti-TNFα antibody) in tobacco BY-2 cells to create a new type of oral biologic to treat inflammatory bowel disease (IBD). Our preliminary data indicate that the plant cells produced oral anti-TNFα biologic was able to ameliorate the disease in the dextran sulfate sodium (DSS)-induced colitis mouse model and induce a reduction of histopathological markers.