Transgenic plants for therapeutic proteins: linking upstream and downstream strategies.

We have described two very different and innovative plant-based production systems--postharvest production and recovery of recombinant product from tobacco leaves using an inducible promoter and oleosin-mediated recovery of recombinant product from oilseeds using a seed-specific promoter. Both base technologies are broadly applicable to numerous classes of pharmaceutical and industrial proteins. As with any emerging technology, the key to success may lie in identifying those products and applications that would most benefit from the unique advantages offered by each system. The postharvest tobacco leaf system appears effective for proteins requiring complex posttranslational processing and endomembrane targeting. Because of the remarkable fecundity and biomass production capacity of tobacco, biomass scale-up is very rapid and production costs are low. Clearly the development of equally cost-effective extraction and purification technologies will be critical for full realization of the commercial opportunities afforded by transgenic plant-based bioproduction. The recovery of protein from tobacco leaves or oleosin-partitioned proteins by oil-body separations represent significant break-throughs for cost-effective commercialization strategies. Additional low-cost, high-affinity separation technologies need to be developed for effective scale-up purification of plant-synthesized recombinant proteins. Clearly successful commercialization of plant-synthesized biopharmaceuticals must effectively link upstream strategies involving gene and protein design with downstream strategies for reproducible GMP-level recovery of bioactive recombinant protein. Both the tobacco and oilseed systems are uniquely designed to address issues of biomass storage, product recovery, quality assurance, and regulatory scrutiny in addition to issues of transgene expression and protein processing.

Purification, characterization, and structural analysis of a plant low-temperature-induced protein.

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.

Inheritance and expression patterns of BN28, a low temperature induced gene in Brassica napus, throughout the Brassicaceae.

Molecular genetics is becoming an important tool in the breeding and selection of agronomically important traits. BN28 is a low temperature induced gene in Brassicaceae species. PCR and Southern blot analysis indicate that BN28 is polymorphic in the three diploid genomes: Brassica rapa (AA), Brassica nigra (BB), and Brassica oleracea (CC). Of the allotetraploids, Brassica napus (AACC) is the only species to have inherited homologous genes from both parental genomes. Brassica juncea (AABB) and Brassica carinata (BBCC) have inherited homologues from the AA and CC genomes, respectively, while Sinapsis arvensis (SS) contains a single homologue from the BB genome and Sinapsis alba (dd) appears to be different from all the diploid parents. All species show message induction when exposed to low temperature. However, differences in expression were noticed at the protein level, with silencing occurring in the BB genome at the level of translation. Results suggest that silencing is occurring in diploid species where duplication may not have occurred. Molecular characterization and inheritance of BN28 homologues in the Brassicaceae may play an important role in determining their quantitative function during exposure to low temperature. Key words : Brassicaceae, BN28, inheritance, polymorphism.

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.

Expression of a Low-Temperature-Induced Protein in Brassica napus.

BN28 is a low-temperature-induced, boiling-soluble protein in Brassica napus. We used antibodies raised against a recombinant BN28 to examine the expression of this protein in cold-acclimating plants and to investigate its relationship to plant freezing tolerance. Changes in the steady-state levels of BN28 protein appear to lag several days behind those of the mRNA. BN28 is first detected on immunoblots after approximately 8 d of exposure to low temperature, and thereafter levels remain stable while plants are maintained at 4[deg]C. Radiolabeling studies indicate that BN28 is synthesized at a relatively low rate. A decline in protein levels is observed soon after returning plants to control temperatures, and little or no protein can be detected after 7 d of deacclimation. The disappearance of the protein precedes a loss in freezing tolerance, suggesting that BN28 is not involved in maintaining plasma membrane integrity. Expression of BN28 is observed primarily in leaves and appears to be low-temperature specific. Quantitative analysis indicated that BN28 accumulates to approximately 82.7 pmol mg-1 total protein in cold-acclimated leaves. This concentration is similar to that reported for two group 2 late-embryogenesis-abundant-like proteins.

Molecular cloning and expression of a turgor-responsive gene in Brassica napus.

During droughting plants activate a number of genes involved in adaptation to water stress. We have isolated one such gene, btg-26, from Brassica napus. Expression of btg-26 is induced in leaf tissue within 72 h of withholding water. At 81% relative water content (RWC), when the plant is just beginning to show signs of wilting, expression is already increased six-fold over levels found in leaf tissue from fully hydrated plants. btg-26 expression reaches a maximum eleven-fold induction at 63% RWC, then transcript levels decrease as RWC continues to drop. btg-26 is also activated in plants exposed to high salinity, low temperature, heat shock and the plant hormone abscisic acid. Analysis of the deduced amino acid sequence revealed similarity to the dehydrogenase family of enzymes. These results suggest that btg-26 encodes a protein whose function may be required early during general osmotic stress in some unknown adaptive metabolic pathway.

Multivariate analyses of polypeptide synthesis in developing maize embryos.

Variation in polypeptide synthesis was examined in developing maize embryos of two inbred and two hybrid genotypes. Multivariate analyses were used to evaluate the variation among two-dimensional, electrophoretic separations of polypeptides. Several features of the data set were revealed. Similar developmental patterns were exhibited by all genotypes and no evidence was obtained for differential rates of development for inbreds and hybrids. The differential synthesis of two subsets of polypeptides during embryo development was observed. The multivariate methods employed in this study were a valuable aid in interpreting the results from a large and complex data set.