Plant Genetic Engineering for Cost-Effective Expression of Therapeutic Proteins
Abstract
Plant expression system offers a versatile platform for producing bioactive molecules, including monoclonal antibodies (mAb) and therapeutic proteins, offering a scalable, rapid, and cost-effective alternative to mammalian cell systems. Aim 1 of this project is to achieve a high expression level of therapeutic proteins. As a proof-of-concept, we selected Rituximab, a commercialized therapeutic mAb, to expressin N. tabacum. We also developed IgG Fc tag fusion constructs for cancer targeting protein production. However, one limitation of plant expression systems is that plant-specific N-glycans, such as β1,2-xylose and core α1,3-fucose, limit clinical translation, altering antibody effector functions eliciting immunogenic responses in humans. Therefore, aim 2 of this project is to modify glycosylation patterns in Nicotiana tabacum via plant genetic engineering that will produce human-like N-glycans, enabling high-yield, cost-effective therapeutic protein expression platforms. We utilized CRISPR/Cas9 multiplex knockout of Xylosyltransferase and Fucosyltransferase genes to eliminate plant-specific N-glycans. Our preliminary results indicate that intact Rituximab (~145 kDa) can successfully assemble in N. tabacum as verified by western blot. We aim to achieve a yield of 10-20 mg/g fresh weight from hydroponically grown N. tabacum. Further, we created IgG Fc tag fusion constructs using a plant expression vector (pCNHP) and a mammalian protein expression vector (pcDNA) for HEK293 mammalian cell expression as a benchmark. The IgG Fc-tag fusion construct will serve as a flexible platform for immunotherapy and peptide ligand expression. As the results for glycoengineering, we successfully established N. tabacum transformation with 44.44% transformation efficiency, verified by the purple callus color from RUBY reporter. The multiplexed CRISPR/Cas9 construct has also been designed. Future efforts focus on affinity purification via Fast Protein Liquid Chromatography (FPLC) and LC/MS identification of protein glycosylation patterns. With a rising global population and expensive therapeutic medicines, this research provides a promising solution to deliver low-cost protein therapeutics.
Start Time
15-4-2026 9:00 AM
End Time
15-4-2026 10:00 AM
Room Number
311
Presentation Type
Oral Presentation
Presentation Subtype
Grad/Comp Orals
Presentation Category
Science, Technology, and Engineering
Student Type
Graduate
Faculty Mentor
Tianhu Sun
Plant Genetic Engineering for Cost-Effective Expression of Therapeutic Proteins
311
Plant expression system offers a versatile platform for producing bioactive molecules, including monoclonal antibodies (mAb) and therapeutic proteins, offering a scalable, rapid, and cost-effective alternative to mammalian cell systems. Aim 1 of this project is to achieve a high expression level of therapeutic proteins. As a proof-of-concept, we selected Rituximab, a commercialized therapeutic mAb, to expressin N. tabacum. We also developed IgG Fc tag fusion constructs for cancer targeting protein production. However, one limitation of plant expression systems is that plant-specific N-glycans, such as β1,2-xylose and core α1,3-fucose, limit clinical translation, altering antibody effector functions eliciting immunogenic responses in humans. Therefore, aim 2 of this project is to modify glycosylation patterns in Nicotiana tabacum via plant genetic engineering that will produce human-like N-glycans, enabling high-yield, cost-effective therapeutic protein expression platforms. We utilized CRISPR/Cas9 multiplex knockout of Xylosyltransferase and Fucosyltransferase genes to eliminate plant-specific N-glycans. Our preliminary results indicate that intact Rituximab (~145 kDa) can successfully assemble in N. tabacum as verified by western blot. We aim to achieve a yield of 10-20 mg/g fresh weight from hydroponically grown N. tabacum. Further, we created IgG Fc tag fusion constructs using a plant expression vector (pCNHP) and a mammalian protein expression vector (pcDNA) for HEK293 mammalian cell expression as a benchmark. The IgG Fc-tag fusion construct will serve as a flexible platform for immunotherapy and peptide ligand expression. As the results for glycoengineering, we successfully established N. tabacum transformation with 44.44% transformation efficiency, verified by the purple callus color from RUBY reporter. The multiplexed CRISPR/Cas9 construct has also been designed. Future efforts focus on affinity purification via Fast Protein Liquid Chromatography (FPLC) and LC/MS identification of protein glycosylation patterns. With a rising global population and expensive therapeutic medicines, this research provides a promising solution to deliver low-cost protein therapeutics.
