Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a 'carrier' for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin...

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Bibliographic Details
Published in:Plant molecular biology 1996, Vol.29 (6), p.1167-1180
Main Authors: Parmenter, D.L, Boothe, J.G, Rooijen, G.J.H. van, Yeung, E.C, Moloney, M.M
Format: Article
Language:English
Subjects:
anticoagulants
Brassica napus
extraction
gene expression
gene transfer
genetic transformation
Hirudo medicinalis
lipid bodies
plant proteins
proteinase inhibitors
purification
recombinant DNA
recombinant proteins
seeds
thrombin
transgenic plants
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Summary:A plant oleosin was used as a 'carrier' for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin 'carrier'. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.
ISSN:0167-4412
1573-5028