Abstract
BackgroundPlants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.ResultsWe have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.ConclusionThese modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.
Highlights
Plants are increasingly being examined as alternative recombinant protein expression systems
Many different plant viruses have been modified to function as expression vectors, Tobacco mosaic virus (TMV)-based vectors express the highest levels of foreign protein in plants (Review [3,4])
Infecting plants with TMV vectors by agroinfiltration A series of plasmids containing Cauliflower mosaic virus (CaMV) 35S promoter (35S) driven TMV vectors, as well as 35S driven versions of the green fluorescent protein gene and the p19 (RNA silencing suppressor) gene from Tomato bushy stunt virus (TBSV) were constructed for this experiment (Figure 1)
Summary
Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. One approach to producing foreign proteins in plants is to generate stable transgenic plant lines. This is a very time consuming and labor intensive process. Plant virus-based vectors allow for the rapid, high level, transient expression of proteins in whole plants (Review [1,2]). Many different plant viruses have been modified to function as expression vectors, Tobacco mosaic virus (TMV)-based vectors express the highest levels of foreign protein in plants (Review [3,4]). TMV's utility as an expression vector has already been well established; TMV vectors have been used to produce of many different kinds of proteins in plants including allergens [5,6], antibodies [7] or antibody fragments [8], and vaccine candidates [9,10]
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