Transgenic plants expressing recombinant vaccine antigens offer several unique advantages over conventional systems, including the large-scale, low cost efficiency associated with modem agriculture. Plants producing recombinant proteins have a diverse range of potential applications, from vaccine production factories, through to disease control and even immunocontraceptive control of pest animal populations.
The aim of this project was to investigate methods for synthesizing high-levels of recombinant proteins in an edible plant, the carrot root. The amount of protein, such as a vaccine antigen, produced per gram of plant tissue, is a major determinant of vaccine production costs, and efficiency. Carrots were chosen as the expression system as they are amenable to transformation, their roots are robust, edible and can be stored at ambient temperature for prolonged periods.
Two approaches were investigated for achieving
high-level expression of transgenes in the carrot root, promoter strength and subcellular targeting of protein products.
Four promoters were investigated, i) the defective embryo and meristem gene (dem) and ii) a-amylase gene (amy) promoters from tomato, iii) the Agrobacterium tumefaciens 2' promoter and iv) the commonly used Cauliflower Mosaic virus (CaMV) 35S promoter as the standard. These were used to drive the uidA (gus) reporter gene, allowing quantitation of transgene expression in both roots and leaves of carrot and tobacco plants for comparison.
To investigate the effects of subcellular targeting on protein accumulation, the green fluorescent protein gene (gfp), under control of the CaMV 35S promoter, was modified to include signal sequences that direct and localize the nascent protein to specific
cellular compartments. These included the i) cytosol, ii) endoplasmic reticulum, iii) plastid and iv) storage vacuoles. The amount of reporter protein produced in transgenic carrot roots was measured.
Transgenic carrots transformed with the CaMV 35S promoter produced the greatest amount of β-glucuronidase (GUS), up to 1.25 µg/mg of extracted soluble protein. Subcellular targeting of the green fluorescent protein (GFP) yielded approximately one hundred fold more protein, up to 122 µg/mg of extracted soluble protein in vacuoles. Distinct patterns of GFP fluorescence in different cellular compartments were seen in the leaves and roots of plants providing proof of correct targeting of GFP.
These results and observations can significantly contribute to future development of plant produced recombinant proteins such as vaccine antigens at increased quantities.