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 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.
Parmenter
D L
DL
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Boothe
J G
JG
van Rooijen
G J
GJ
Yeung
E C
EC
Moloney
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001937:Brassica / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D006629:Hirudins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D007865:Leeches; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012639:Seeds; D012689:Sequence Homology, Nucleic Acid; D013917:Thrombin / Q000037:antagonists & inhibitors; D014162:Transfection
D029681:Arabidopsis Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D006629:Hirudins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C079353:oleosin protein, Arabidopsis; D013917:Thrombin