Optimization of the human colorectal carcinoma antigen GA733-2 production in tobacco plants

• Published in: Plant biotechnology reports 2021, 15(1), , pp.55-67
• Main Authors: Park, Se Hee, Ji, Kon-Young, Kim, Hyun Min, Ma, Sang Hoon, Park, Seo Young, Do, Ju Hui, Oh, Doo-Byoung, Kang, Hyung Sik, Shim, Jae Sung, Joung, Young Hee
• Format: Article
• Language: English
• Subjects: Agriculture; Antigens; Biological activity; Biomedical and Life Sciences; Biotechnology; Cancer; Cancer vaccines; Cassettes; Cell Biology; Colorectal cancer; Colorectal carcinoma; Fc receptors; Gene expression; Glycosylation; Life Sciences; Nicotiana benthamiana; Optimization; Original; Original Article; Plant Biochemistry; Plant Sciences; Plants; Proteins; Stability analysis; T-DNA; Tobacco; Transgenic plants; Vaccine development; Vaccines; 농학
• ISSN: 1863-5466; 1863-5474
• DOI: 10.1007/s11816-020-00657-y

The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its significant importance for colorectal vaccine development, low efficiency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that affect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA affected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species ( Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana ) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana . Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also significantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efficient production of functional rGA733-2 protein in tobacco system.