title: Extremely high-level and rapid transient protein production in plants without the use of viral replication.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom.
authors.forename: Frank
authors.initials: F
authors.lastname: Sainsbury
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
mesh_terms: D001483:Base Sequence; D003062:Codon; D004742:Enhancer Elements, Genetic; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D014779:Virus Replication
chemical_list: D003062:Codon; D010940:Plant Proteins; D012333:RNA, Messenger
references.citation: J Biotechnol. 2007 Jan 10;127(3):335-47
references.pmid: 16959350
references.citation: Plant Physiol. 2007 Dec;145(4):1232-40
references.pmid: 17720752
references.citation: Plant Biotechnol J. 2006 Mar;4(2):183-93
references.pmid: 17177795
references.citation: Plant Cell Rep. 2003 Sep;22(2):117-21
references.pmid: 12845471
references.citation: Plant Biotechnol J. 2008 Feb;6(2):189-201
references.pmid: 17979949
references.citation: J Virol Methods. 2006 Jan;131(1):10-5
references.pmid: 16112207
references.citation: J Virol. 1993 Jun;67(6):3660-4
references.pmid: 8497075
references.citation: Virology. 1993 Aug;195(2):377-86
references.pmid: 8337819
references.citation: AIDS Res Hum Retroviruses. 1994 Apr;10(4):359-69
references.pmid: 7520721
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 3;103(40):14701-6
references.pmid: 16973752
references.citation: Vaccine. 2005 Mar 7;23(15):1784-7
references.pmid: 15734041
references.citation: Plant Cell. 1997 Jun;9(6):859-68
references.pmid: 9212462
references.citation: Virus Res. 2006 Jul;119(1):52-62
references.pmid: 16325949
references.citation: Transgenic Res. 1995 Jul;4(4):288-90
references.pmid: 7655517
references.citation: Virology. 1989 Sep;172(1):311-20
references.pmid: 2773321
references.citation: Trends Biotechnol. 2003 Dec;21(12):570-8
references.pmid: 14624867
references.citation: Biochimie. 1993;75(8):741-7
references.pmid: 8286445
references.citation: Nat Rev Genet. 2003 Oct;4(10):794-805
references.pmid: 14526375
references.citation: Plant J. 2003 Mar;33(5):949-56
references.pmid: 12609035
references.citation: Plant Biotechnol J. 2008 Jan;6(1):82-92
references.pmid: 17986176
references.citation: Genes Dev. 1998 Aug 1;12(15):2293-304
references.pmid: 9694795
references.citation: Vaccine. 2005 Mar 7;23(15):1788-92
references.pmid: 15734042
references.citation: Mol Cell Biol. 2000 Dec;20(23):8635-42
references.pmid: 11073965
references.citation: Virology. 1993 Apr;193(2):672-9
references.pmid: 8460480
references.citation: Virus Res. 2005 Feb;107(2):141-9
references.pmid: 15649560
references.citation: Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1114-24
references.pmid: 17001089