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

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Abstract

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.

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Acknowledgements

This work was supported by a grant from the New breeding technologies development Program (Project No. PJ01487401), and the Next-Generation BioGreen21 Program (Project No. PJ01389401), Rural Development Administration, Republic of Korea.

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  1. School of Biological Sciences and Technology, Chonnam National University, Gwangju, 61186, Korea

    Se Hee Park, Hyun Min Kim, Sang Hoon Ma, Seo Young Park, Ju Hui Do, Hyung Sik Kang, Jae Sung Shim & Young Hee Joung

  2. Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, 34054, Korea

    Kon-Young Ji

  3. Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Korea

    Doo-Byoung Oh

  4. Department of Biosystems and Bioengineering, University of Science and Technology (UST), Daejeon, 34113, Korea

    Doo-Byoung Oh

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  1. Se Hee Park
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Park, S.H., Ji, KY., Kim, H.M. et al. Optimization of the human colorectal carcinoma antigen GA733-2 production in tobacco plants. Plant Biotechnol Rep 15, 55–67 (2021). https://doi.org/10.1007/s11816-020-00657-y

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