PRODUCTION OF BIOLOGICALLY-ACTIVE HIRUDIN IN PLANT SEEDS USING OLEOSIN PARTITIONING

A plant oleosin was used as a 'carrier' for the production of the leec h anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusi on protein was expressed and accumulated in seeds. Seed-specific expre ssion of the oleosin-hirudin fusion mRNA was directed via an Arabidops is oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed protei ns by flotation centrifugation. Recombinant hirudin was localized to t he surface of oil bodies as determined by immunofluorescent techniques . The oleosin-hirudin fusion protein accumulated to ca. 1% of the tota l seed protein. Hirudin was released from the surface of the oil bodie s using endoprotease treatment. Recombinant hirudin was partially puri fied through anion exchange chromatography and reverse-phase chromatog raphy. Hirudin activity, measured in anti-thrombin units (ATU), was ob served in seed oil body extracts, but only after the proteolytic relea se of hirudin from its oleosin 'carrier'. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. Th is activity demonstrated linear dose dependence. The oleosin fusion pr otein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for p urification of the desired polypeptide.