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.

结直肠癌相关蛋白GA733-2是开发植物源性结直肠癌疫苗的代表性候选蛋白之一。尽管 GA733-2 对结直肠疫苗的开发具有重要意义，但 GA733-2 的低生产效率限制了其广泛应用。为了提高 GA733-2 在植物中的生产力，我们在此测试了影响重组 GA733-2 (rGA733-2) 和融合到片段可结晶 (Fc) 结构域 (rGA733-Fc) 蛋白的 rGA733 表达的多种因素。当 pBINPLUS 载体系统用于烟草植物中的瞬时表达时，rGA733-2 和 rGA733-Fc 蛋白得到高表达。此外，rGA733-2之间的间隔长度T-DNA和左边界影响rGA733蛋白的表达。使用根癌农杆菌菌株（C58C1、LBA4404 和 GV3101）和烟草物种（Nicotiana tabacum cv. Xanthi nc 和Nicotiana benthamiana ）的各种组合的瞬时表达分析表明，rGA733-2 和 rGA733-Fc 蛋白的更高积累是通过组合A. tumefaciens LBA4404 和Nicotiana benthamiana. 通过引入 rGA733-2 和 rGA733-Fc 表达盒产生的转基因植物也显着积累了相应的重组蛋白。通过进一步的体外测定、蛋白质印迹和 N-糖基化分析评估植物来源的 rGA733 和 rGA733-Fc 的生物活性和稳定性。总的来说，我们在此建议在烟草系统中有效生产功能性 rGA733-2 蛋白的最佳条件。