Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing

Abstract

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.

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…An example is N. benthamiana, an Australian native plant that has found increasing application for recombinant and transient foreign protein synthesis in vitro [29,38,39]. This plant is known for its hypersusceptibility to infection by plant viruses.…”

“…This approach restricts transformation and protein expression to only certain stages of cell culture, thereby avoiding the reductions in expression levels that occur during multiple cell divisions due to mutations or genetic re-arrangements. Co-expression of viral proteins that suppress post-transcriptional gene silencing has also been used to minimise gene silencing effects during transient production of foreign proteins in plant suspension cultures [29,39]. In vivo, the local spread of RNA silencing signals occurs by symplastic transfer between plant cells through plasmodesmata [53].…”

“…Tobacco cell lines such as BY-2 (bright yellow 2) and NT-1 (Nicotiana tabacum 1) are readily available, are amenable to Agrobacteriummediated genetic transformation, and have been optimised for rapid cell growth. Other host species applied for in vitro foreign protein synthesis include rice [24], soybean [25], tomato [26], carrot [18,27], Arabidopsis [28], Medicago truncatula [28] and N. benthamiana [29]. When in vitro plant cultures are used for protein production, the growth habit of the host species, such as whether it grows upright, is a high biomass producer in agronomic terms, or is suitable for mechanised agriculture, is no longer an important consideration.…”

“…Several systems have been developed for transient expression of foreign proteins in plant suspension and hairy root cultures based on viral vectors or agrobacterial co-culture [29,39,[50][51][52]. This approach restricts transformation and protein expression to only certain stages of cell culture, thereby avoiding the reductions in expression levels that occur during multiple cell divisions due to mutations or genetic re-arrangements.…”

Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture

“…The pEAQ-HT vector (Sainsbury et al, 2009), based on this system, includes a gene encoding the tomato bushy stunt virus (TBSV) protein P19 (Scholthof, 2007) on the same T-DNA as the target gene. P19 can efficiently suppress posttranscriptional gene silencing (PTGS) that commonly limits the productivity of plant expression systems (Boivin et al, 2010). The pEAQ-HT vectors have been extremely useful for transient expression in Nicotiana benthamiana leaves (Sainsbury et al, 2010), but there is no report of their use to enhance expression in plant suspension cell cultures.…”