High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

Abstract

SummaryWe constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three-to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.

In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants

“…The current strong trend is the application of disaggregated virus vector systems for transient expression in, almost exclusively, N. benthamiana, via agroinfiltration (Giritch et al, 2006;Regnard et al, 2010;Sainsbury et al, 2010). These systems provide for (1) quick, high-level expression and (2) rapid development from design and assembly of an expression cassette to expressed protein, the rapid response concept being of particular relevance to the deployment of vaccines for new infectious diseases.…”

DNA Replicons for Plant Genome Engineering

“…We chose to deconstruct the mild strain of the BeYDV (Halley-Stott et al, 2007) because it had previously been used to express heterologous proteins in plant cells (Regnard et al, 2010). BeYDV replication requires three viral elements: the cis-acting LIR, the short intergenic region (SIR), and the trans-acting replicationinitiation protein (Rep) and RepA.…”

“…Lack of cell-to-cell movement can be compensated using Agrobacterium or particle bombardment to deliver viral vectors to cells. This approach has been successfully used to generate replicons based on the bean yellow dwarf virus (BeYDV) for expression of reporter proteins, vaccine proteins and monoclonal antibodies (Mor et al, 2003;Zhang and Mason, 2006;Huang et al, 2009Huang et al, , 2010Regnard et al, 2010). Currently, there is no obvious upper size limit for replicons that do not move from cell to cell (Laufs et al, 1990;Matzeit et al, 1991;Shen and Hohn, 1994;Huang et al, 2009).…”

Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants

“…Interestingly, when C1/C2 genes were present in the replicon, co-delivery of a p19 expression cassette had no discernable effect on expression of HBcAg. Rybicki and coworkers 20 have also utilized replicons that contain the C1/C2 genes from a "mild" strain of BeYDV (BeYDV-m). The replicon is very similar to that described above, 19 and produced substantial enhancement of expression over that obtained with a nonreplicating vector for three different genes.…”