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Published on September 5, 2008; 10.1104/pp.108.126284

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Received July 11, 2008
Accepted September 2, 2008

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication

Frank Sainsbury and George P. Lomonossoff *

Dept. of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

* Corresponding author; email: george.lomonossoff{at}bbsrc.ac.uk.

Plant-based over-expression of heterologous proteins has attracted much interest and development in recent years. To date the most efficient vectors are based on RNA virus-derived replicons. A system based on a disabled version of Cowpea mosaic virus (CPMV) RNA-2 has been developed, which overcomes limitations on insert size and introduces biocontainment. This system involves positioning a gene of interest between the 5' leader sequence and 3' UTR of RNA-2, thereby emulating a presumably stable mRNA for efficient translation. Thus far the sequence of the 5'UTR has been preserved to maintain the ability of the modified RNA-2 to be replicated by RNA-1. However, high-level expression may be achieved in the absence of RNA-1-derived replication functions using Agrobacteria-mediated transient transformation. To investigate those features of the 5' UTR necessary for efficient expression we have addressed the role of two AUG codons found within the 5' leader sequence upstream of the main initiation start site. Deletion of in-frame start codons upstream of the main translation initiation site led to a massive increase in foreign protein accumulation. By 6 days post-infiltration, a number of unrelated proteins, including a full size IgG and a self-assembling virus-like particle, were expressed to greater than 10 and 20% of total extractable protein, respectively. Thus this system provides an ideal vehicle for high-level expression that does not rely on viral replication of transcripts.




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