Production of biologically active hirudin in plant seeds using oleosin partitioning

Abstract

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.

Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain

“…It is likely that membrane topology and the hydrophobicity of the H domain favors transition into a region of oil in the ER membrane from which oil body formation can occur (18 -20). Oleosins are also used as carriers for recombinant protein production in seeds (21,22). Thus, the topology of this protein has a significant impact on the potential uses of oleosin-based production systems.…”

Highly efficient production of human interferon-.ALPHA. by transgenic cultured rice cells

“…Recently, many biopharmaceutical researchers have been actively developing vaccines and therapeutic proteins utilizing transgenic plants (Kumagai et al 1993, Parmenter et al 1995, Dieryck et al 1997, Tacket and Mason 1999. In this study, we demonstrated that IFN, a biologically active human protein could be obtained in a stable manner from cultivated calli using a simple inorganic liquid medium.…”

Production of biologically active human interferon-.ALPHA. in transgenic rice

“…Recently, many biopharmaceutical investigators have begun to actively develop vaccines and therapeutic proteins utilizing transgenic plants (Kumagai et al 1993;Parmenter et al 1995;Dieryck et al 1997;Tacket and Mason 1999;Ma et al 2005). Considering the applications for oral administration and immunization, cereals such as rice will be utilized to produce these proteins.…”