Utility of the P19 suppressor of gene‐silencing protein for production of therapeutic antibodies in Nicotiana expression hosts

Abstract

SummaryTo study how the P19 suppressor of gene-silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co-expressed with P19 in an RNAibased glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102-106) containing different 5′ and 3′ UTRs, designated as vector sets 102-106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15-fold (to about 2.3% of TSP) when co-expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co-expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I-64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19-induced responses.

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

“…On day 5 post infiltration, some slight discoloration was detected mostly in the younger leaves (Figure 8C). Notably, p19 infiltrated tissue yielded higher amounts of GFP compared to mock-infiltrated tissue, contrary to the results obtained with p19 from TBSV [48]. …”

“…It has been previously shown that p19 from tomato bushy stunt virus (TBSV) induces a hypersensitive response in tobacco cv. I64 infiltrated tissue which not only reduces the biomass yield of tobacco plants but also negatively affects the accumulation levels of co-expressed proteins starting at 3 dpi [47,48]. We monitored infiltrated 81V9 and I64 leaves from 2 to 5 dpi, and did not observe any discoloration or other signs of necrosis before 5 dpi.…”

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

“…Wild-type (P35S:MSL10-GFP), phosphomimetic (P35S:MSL10 D -GFP), or alanine substitution (P35S:MSL10 A -GFP) versions of MSL10 were transiently overexpressed in tobacco leaves via infiltration with Agrobacterium tumefaciens transformed with the binary vector pK7FWG2 (Karimi et al, 2002) harboring the appropriate MSL10 sequences. A plasmid encoding the p19 gene of tomato bushy stunt virus, which is often used in transient expression systems to inhibit transgene silencing (Voinnet et al, 2003;Waadt and Kudla, 2008;Garabagi et al, 2012), was coinfiltrated with each construct. Three days after infiltration, successful expression was confirmed through GFP fluorescence ( Figure 3A, top row).…”

Antibodies from plants for bionanomaterials

“…Plantspecific N-glycosylation, an important productspecific and application-specific issue for antibodies made in plants, has been addressed using classical genetic engineering approaches [163][164][165][166] as well as more recent genome editing technologies. 167 A number of auxiliary technologies have been used to address post-transcriptional gene silencing, 168 target protein folding and proteolysis, [169][170][171] and downstream processing strategies for extraction, clarification, and purification. 172 Manufacturing capacity is being built at several international locations (Table 2) and models predicting the manufacturing costs are being refined.…”