Tobacco mosaic virus and the virescence of biotechnology

Abstract

There is a growing realization that a modern combination of molecular biology and agriculture will provide a photosynthetic basis for the biosynthesis of an increasing variety of complex and valuable molecules. This`greening' of biotechnology may impact on the global environment in many bene¢cial ways, but will perhaps have its most signi¢cant impact on human health. In the past decade, the capacity to use plants as an expanded source of therapeutics has grown through the accelerated development of e¡ective viral transfection vectors for gene transfer to cultivated crops. Recombinant vectors based on tobacco mosaic virus (TMV) and other members of the Tobamovirus genus are now used to transfect commercially meaningful quantities of plant biomass cultivated in enclosed greenhouses and multiacre ¢elds. Viral RNA promoters are e¡ectively manipulated for the synthesis of recombinant messenger RNAs in whole plants. Chimeric plant virus and virus-like particles are designed for peptide production and display from recombinant structural protein^gene fusions. Gene functions are assessed and modi¢ed by either virus-mediated expression or cytosolic inhibition of expression at the RNA level. Recombinant virus populations, propagated by inoculating plants with infectious RNA transcripts or recombinant virions, have proved to be genetically stable over product-manufacturing cycles. Large volumes of highly puri¢ed protein products isolated from transfected foliage conform reproducibly to the speci¢cations required for well-characterized biologics. In some cases, they exceed the speci¢c activities of molecules puri¢ed from alternative recombinant and native sources. The resulting products are then formulated according to the developing national regulatory guidelines appropriate for agriculture-based manufacturing. Each of these innovations was ¢rst realized by researchers using clones of tobamovirus genes and recombinant genomes. This progress is founded on the heritage of a century of fundamental TMV research.

In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium

“…Theoretically, expression systems based on viral vectors hold a great promise (for reviews, see refs. [1][2][3]. The tobacco mosaic virus (TMV)-based vectors developed by the company Large Scale Biology (4) have been successfully developed into a functional genomics engine with the highest possible throughput (5,6), and similar vectors are being used for commercial-scale production of pharmaceutical proteins, some of which are currently undergoing clinical trials.…”

Production of Recombinant Proteins from Transgenic Crops

“…Finally, secondary metabolites such as neonicotine (anabasine) and nicotine must usually be removed early in the purification process (Theisen, 1999). Vacuum infiltration of green leaves with buffer, followed by mild centrifugation has been reported for the purification of a-galactosidase A (Turpen, 1999). However, it is not clear if this method can handle high throughputs of biomass (Doran, 2000).…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…Sequences in the tobamovirus genome function as subgenomic promoters regulating the production of subgenomic RNAs. The virus movement protein MP and CP are translated from two separate, but co-terminal, subgenomic RNAs, with the CP being among the most abundant protein and RNA produced in the infected cell [46]. In a tobamovirus infected plant there are several milligrams of CP produced per gram of infected tissue.…”

“…Several reviews have provided surveys of these products [12,14]. In general, expression results were obtained in Nicotiana hosts and proteins were extracted from leaf tissues using total homogenization and clarification methods or leaf infiltration and isolation of interstitial fluids [46,47]. Several products have been purified using differential separation and standard chromatographic separations and tested in various model systems of human disease.…”

“…This movement produces the systematic infection and the foreign gene is expressed in all cells that express other virus protein products. Within the cell, the foreign protein is deposited in the site dictated by its protein sequence, either naturally or purposely engineered [12,46].…”