title: Optimization of Ammonium Sulfate Concentration for Purification of Colorectal Cancer Vaccine Candidate Recombinant Protein GA733-FcK Isolated from Plants.
abstract: A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25-30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. Thus, the objective in this study is to optimize the conditions for plant-derived protein purification procedures. Various ammonium sulfate concentrations (15-80%) were compared to determine their effects on TSPs yield. With 50% ammonium sulfate, the yield of precipitated TSP was the highest, and that of the plant-derived colorectal cancer-specific surface glycoprotein GA733 fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733(P)-FcK) protein significantly increased 1.8-fold. SDS-PAGE analysis showed that the purity of GA733(P)-FcK protein band appeared to be similar to that of an equal dose of mammalian-derived GA733-Fc (GA733(M)-Fc). The binding activity of purified GA733(P)-FcK to anti-GA733 mAb was as efficient as the native GA733(M)-Fc. Thus, the purification process was effectively optimized for obtaining a high yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery rate of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising solution for production of recombinant GA733-Fc protein in plants.
authors.affiliation: Therapeutic Protein Engineering Lab, Department of Medicine, College of Medicine, Chung-Ang University Seoul, South Korea.
authors.forename: Se-Ra
authors.initials: SR
authors.lastname: Park
authors.affiliation: Therapeutic Protein Engineering Lab, Department of Medicine, College of Medicine, Chung-Ang University Seoul, South Korea.
authors.forename: Chae-Yeon
authors.initials: CY
authors.lastname: Lim
authors.affiliation: Therapeutic Protein Engineering Lab, Department of Medicine, College of Medicine, Chung-Ang University Seoul, South Korea.
authors.forename: Deuk-Su
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Therapeutic Protein Engineering Lab, Department of Medicine, College of Medicine, Chung-Ang University Seoul, South Korea.
authors.forename: Kisung
authors.initials: K
authors.lastname: Ko
keywords: GA733-Fc; ammonium sulfate; colorectal cancer; recombinant vaccine; transgenic plant