TRANSGENIC BRASSICA-CARINATA AS A VEHICLE FOR THE PRODUCTION OF RECOMBINANT PROTEINS IN SEEDS

Hirudin, a blood anticoagulant protein from leeches, and P-glucuronida se were produced in Brassica carinata Braun (Ethiopian mustard) seeds using oleosin as a carrier. Cotyledonary petioles were infected with A grobacterium strains containing oleosin-glucuronidase (pCGNOBPGUS-A) o r oleosin-hirudin (pCGN-OBHIRT) constructs. Polymerase chain reaction and neomycin phosphotransferase II enzyme assays confirmed the presenc e of the fusion genes in plants regenerating under selection. The fusi on polypeptides were correctly expressed and targeted to the oil-bodie s of the seeds with high fidelity (ca. 90%). Recombinant protein was p urified from all other cellular protein by a simple flotation process and cleaved from oil-bodies using the endoprotease, Factor Xa. Hirudin activity was measured using a colorimetric thrombin inhibition assay and an activity in the range of 0.2-0.4 antithrombin units per milligr am of oil-body protein was detected. B. carinata offers an attractive alternative for the production of recombinant proteins using oleosin t echnology.