| This thesis presents a study on the use of oil body proteins, or oleosins, as "carriers" for seed-localized recombinant proteins in plants. Oleosins are unique proteins which decorate the surface of oil bodies in seeds. They are co-translated on the endoplasmic reticulum and target specifically to oil bodies. Starting with a gene encoding an oleosin derived from Arabidopsis, a model system for protein production in seeds was created. The model protein used was hirudin, a pharmaceutically valuable blood anticoagulant originally derived from medicinal leeches.;Genetic constructs comprising a synthetic, mature hirudin variant 2 cDNA fused, in-frame, to the 3;The above experiments clearly demonstrated that oleosins could be used as fusional "carriers" for the production of biologically active hirudin. In an effort to enhance expression and protein accumulation of the oleosin-hirudin fusion gene/protein, the above construct was modified. A longer Arabidopsis oleosin promoter was used to drive the expression of a B. napus oleosin-hirudin variant 1 fusion gene harbouring, or lacking, an alfalfa mosaic virus RNA4 (AMV) leader sequence. Preliminary results indicate that the fusion protein accumulated to higher levels.;In a third set of experiments, three constructs, having 1, 2, or 4 hirudin-encoding concatameric sequences fused, in-frame, to a B. napus cDNA were created. Analysis of seed extracts from transformants harbouring these constructs showed that the fusion protein accumulated and targeted to the oil body. |
