Bio-P2 Research Unit, Dept. of Chemical Engineering, École Polytechnique de Montréal, Centre-ville Station, Montréal, Québec, Canada H3C 3A7.
Biotechnol Prog. 2010 Nov-Dec;26(6):1534-43. doi: 10.1002/btpr.485.
Two distinct transient expression approaches were compared with assess the impact of the viral suppressor p19 on a recombinant protein production performed in Nicotiana benthamiana suspension culture. A parental N. benthamiana cell line was transiently transformed with either an Agrobacterium containing a gene construct for a murine IgG1 (R514) or concurrently with two Agrobacteria containing R514 or p19. In addition, a stably transformed N. benthamiana cell line that constitutively expresses p19 was transformed with R514-containing Agrobacterium. The parental N. benthamiana cell line that had been co-cultivated with both p19 and R514 achieved the highest yield of IgG1 (1.06 mg IgG1/kg FW; 0.024% TSP) compared with that obtained without p19 (0.61 mg IgG1/kg FW; 0.014% TSP). The N. benthamiana cell line that had been stably transformed with p19 only reached 0.25 mg IgG1/kg FW (0.009% TSP) when co-cultured with R514-containing Agrobacterium. Dual agroinfiltration of N. benthamiana leaves with p19 and R514 was also performed to assess for Agrobacteria efficiencies and 147.7 mg IgG1/kg FW were obtained. Therefore, our results demonstrate that transient co-transformation of plant cell suspension culture with two transformation vectors is feasible and that the use of the viral suppressor of silencing p19 significantly raises the production of the protein of interest.
两种不同的瞬时表达方法被比较,以评估病毒抑制剂 p19 对在烟草悬浮培养细胞中进行的重组蛋白生产的影响。瞬时转化亲本烟草细胞系,使用含有鼠 IgG1(R514)基因构建体的农杆菌或同时使用两种含有 R514 或 p19 的农杆菌。此外,使用含有 R514 的农杆菌转化稳定转化 p19 的烟草细胞系。与未共转化 p19 的细胞系相比,与 p19 和 R514 共培养的亲本烟草细胞系实现了最高 IgG1 产量(1.06 mg IgG1/kgFW;0.024%TSP)。当与含有 R514 的农杆菌共培养时,仅稳定转化 p19 的烟草细胞系达到 0.25 mg IgG1/kgFW(0.009%TSP)。还进行了 p19 和 R514 的双农杆菌叶浸润,以评估农杆菌效率,获得了 147.7 mg IgG1/kgFW。因此,我们的结果表明,瞬时共转化植物悬浮细胞培养物的两种转化载体是可行的,并且使用沉默抑制病毒抑制剂 p19 可显著提高目标蛋白的产量。