This Article Full Text Full Text (PDF) All Versions of this Article: 148/3/1212    most recent pp.108.126284v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Sainsbury, F. Articles by Lomonossoff, G. P. Search for Related Content PubMed PubMed Citation Articles by Sainsbury, F. Articles by Lomonossoff, G. P. Agricola Articles by Sainsbury, F. Articles by Lomonossoff, G. P. Related Collections Vector Systems for Plant Research and Biotechnology

BREAKTHROUGH TECHNOLOGIES

Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication¹

Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom

Plant-based overexpression of heterologous proteins has attracted much interest and development in recent years. To date, the most efficient vectors have been based on RNA virus-derived replicons. A system based on a disabled version of cowpea mosaic virus 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' untranslated region (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 Agrobacterium-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 an in-frame start codon upstream of the main translation initiation site led to a massive increase in foreign protein accumulation. By 6 d postinfiltration, a number of unrelated proteins, including a full-size IgG and a self-assembling virus-like particle, were expressed to >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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: George P. Lomonossoff (george.lomonossoff{at}bbsrc.ac.uk).

www.plantphysiol.org/cgi/doi/10.1104/pp.108.126284

^* Corresponding author; e-mail george.lomonossoff{at}bbsrc.ac.uk.

Received July 11, 2008; accepted September 2, 2008; published September 5, 2008.