| With recent advances in biotechnology, plants have been genetically engineered to manufacture proteins for applications in medicine, industry and the basic sciences. Plants offer advantages as foreign protein factories because of ease of handling, cost-effectiveness, and ease of scale-up due to existing agricultural infrastructure. Since plant-produced recombinant proteins are designed for use in industry, high protein yields are desirable. Methods for increasing protein yields include constitutive protein expression, tissue specific expression and subcellular targeting. The objective of this project was to determine if vacuolar targeting sequences that normally direct proteins to tuber vegetative storage vacuoles or leaf lytic or neutral vacuoles would target proteins to seed protein storage vacuoles, since they appear to be analogous structures. We were interested in foreign protein accumulation in seeds because seeds produce and stably store high levels of protein over a long time period. This study utilized GFP fused to various vacuolar targeting signals and driven by the enhanced CaMV 35S promoter for expression in Arabidopsis thaliana plants. Targeting sequences from sweet potato sporamin, potato cathepsin inhibitor, tobacco chitinase, potato 22kDa protein, and Brazil nut albumin were assayed for targeting function and subcellular fusion protein localization in leaves and seeds. Based on fluorescence microscopy and transmission electron microscopy of immunogold labeled samples, we demonstrated that regardless of the source, these sequences all targeted GFP to leaf vacuoles and the seed protein storage vacuole matrix. Also, vacuolar signals normally located at the N-terminus were functional at the C-terminus of the fusion protein. GFP accumulation was not significantly different among the different vacuolar sorting determinants. GFP was stably expressed and accumulated to 0.2% total soluble protein. Since practical applications require high foreign protein yields, this information may be useful in maximizing plant recombinant protein production. |
