Abstract
Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5′ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower Agrobacterium concentrations than non-replicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5′ UTR does not.
Highlights
Plant-based expression systems offer many potential advantages over traditional systems, including safety, speed, versatility, scalability, and cost (Gleba et al, 2014; Tusé et al, 2014; Chen and Davis, 2016; Nandi et al, 2016)
In the bean yellow dwarf virus (BeYDV) expression system (Figure 1A), production of Rep/RepA leads to excision, circularization, and replication of any gene expression cassette flanked by the cis-acting long intergenic region (LIR)
We showed that a Rep/RepA-supplying vector could be delivered in trans to amplify a replication-deficient BeYDV containing the LIRs but lacking Rep/RepA (Huang et al, 2009)
Summary
Plant-based expression systems offer many potential advantages over traditional systems, including safety, speed, versatility, scalability, and cost (Gleba et al, 2014; Tusé et al, 2014; Chen and Davis, 2016; Nandi et al, 2016). A plant-based transient expression system has been developed which uses the replication machinery from the geminivirus bean yellow dwarf virus (BeYDV) to substantially increase transgene copy number in the plant nucleus, with a subsequent increase in transcription of the target gene (Huang et al, 2009, 2010) This system has been used to produce high levels of vaccine antigens and pharmaceutical proteins in Nicotiana benthamiana leaves (Phoolcharoen et al, 2011; Lai et al, 2012; Moon et al, 2014; Kim et al, 2015; Diamos et al, 2016; Diamos and Mason, 2018). The factors contributing to cell death in the BeYDV system have not been thoroughly investigated
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