Transgenic plants have significant potential in the bioproduction of complex human therapeutic
proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating
protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human
proteins with commercial potential as human
pharmaceuticals. Human
protein C (hPC), a highly processed serum
protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages,
disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of
protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal
enzyme glucocerebrosidase (hGC) in transgenic tobacco. This
glycoprotein has significant commercial potential as replacement
therapy in patients with
Gaucher's disease. Regular
intravenous administration of modified
glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with
Gaucher's disease. However, the
enzyme is expensive (dubbed the "world's most expensive
drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human
enzymes. Transgenic tobacco plants were generated that contained the human
glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in
plant extracts by
enzyme activity assay and immunologic cross-reactivity with anti-hGC
antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in
crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual
therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human
proteins of
biologic and
pharmaceutical interest.