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.

Viviparous-5 encodes phytoene desaturase, an enzyme essential for abscisic acid (ABA) accumulation and seed development in maize.

The role of synthesis in the regulation of abscisic acid accumulation was investigated in the developing maize seed. To do this, expression and regulation of the abscisic acid biosynthetic enzyme phytoene desaturase were examined. Comparison of the gene sequence encoding phytoene desaturase and its transcript in the wild-type and viviparous-5 mutant showed that the mutant gene contains multiple insertions and deletions, resulting in the synthesis of a larger transcript. In addition, the 55-kDa phytoene desaturase protein was not detectable in the viviparous-5 mutant, indicating that this phenotype results from a mutation at the phytoene desaturase locus. Levels of phytoene desaturase transcript and protein were compared to abscisic acid levels during development to determine whether phytoene desaturase might regulate abscisic acid accumulation. In the endosperm, transcript levels were initially high and declined during late maturation and dormancy, while protein levels remained high throughout development. In the embryo, transcript levels were low and constant, while protein levels declined. Both temporal and tissue-specific expression of phytoene desaturase were unrelated to abscisic acid levels. An abscisic acid mutant (viviparous-2) deficient in phytoene desaturation was used to determine whether the wild-type protein encoded by Viviparous-2 regulates phytoene desaturase. Phytoene desaturase transcript and protein levels were compared in wild-type and viviparous-2 mutant embryos and endosperm. Normalized levels of phytoene desaturase were similar in wild-type and mutant tissues, suggesting that the wild-type Viviparous-2 protein does not regulate phytoene desaturase transcript or protein levels.

Bioproduction of human enzymes in transgenic tobacco.

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.

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in the Endosperm of Maize vivipary Mutants.

During seed maturation the levels of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) decrease while the levels of the isoprenoid compund abscisic acid (ABA) increase. In the present study, we demonstrate that HMGR specific activity is inversely correlated with endogenous ABA levels in Zea mays endosperm during seed development. HMGR specific activity and ABA levels were measured in the endosperm of the maize vivipary mutuants vp2, vp5, and vp7, which are defective in ABA biosynthesis, and vp1, which is defective in an ABA response element. Reduced ABA levels were observed in the endosperm of vp2, vp5, and vp7, whereas HMGR activity levels were higher compared with wild-type sibling endosperm activity. HMGR activities were increased by as much as 37% (vp2), 45% (vp5), and 58% (vp7) in the mutants. Endosperm HMGR activity in the vp1 mutant was also increased (41%) relative to wild-type siblings, even though vp1 does not have reduced ABA levels. In addition, exogenous ABA inhibits HMGR activity 34 to 50% in maize roots. These results sugggest the HMGR activity levels during seed development are regulated via a Vp1-dependent signal transduction pathway that is affected by the reduced ABA content of vp2, vp5, and vp7 endosperm.

Characterization of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity during Maize Seed Development, Germination, and Seedling Emergence.

Many isoprenoid compounds are necessary for growth and development of the seed and seedling. The first committed step in the biosynthesis of isoprenoid compounds is the conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate. This study shows that the specific activity of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is developmentally regulated during Zea mays seed development and seedling emergence. The highest activities were observed in seed development during stages of rapid mitotic divisions: 10 to 12 d after pollination in the endosperm (216.1 units) and embryo (140.2 units). During stages of maturation, the endosperm HMGR activity decreases to one-fifth the maximal activity, and the embryo activity remains high at one-half the maximal activity. Both the endosperm and embryo HMGR activities decrease to a basal level (2.0 units) in the desiccated seed. At approximately 48 h after imbibition, the embryo HMGR activity significantly increases to 5.1 units. In seeds germinated under white light, root HMGR activity is 2- to 4-fold higher than shoot activity. In seeds germinated in the dark, both root and shoot HMGR activities are 1- to 5-fold higher relative to activities in light-grown seeds.

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.

Organ-specific expression of maize Adh1 is altered after a Mu transposon insertion.

A new, unstable, organ-specific Adh1 mutant was isolated from a Robertson's mutator line by germinating kernels under partial anaerobic conditions. Families of kernels which showed segregation of a conditional anaerobic lethal phenotype were identified. One mutant, Adh1-3F1124, was shown to express approximately 6% normal levels of ADH1 in seed and anaerobically treated seedlings but expresses normal levels of ADH1 in pollen, the male gametophyte. The ADH1 polypeptide encoded by the mutant allele was found to be indistinguishable from that encoded by the Adh1-3F progenitor but its message levels were lower in seed and seedlings. Robertson's mutator lines are known to carry Mu transposons that cause increased mutation rates. Genomic Southern analysis of Adh1-3F1124 and Adh1-3F showed the presence of a 1.85 kbp insertion at the 5' region of Adh1. Comparison of the DNA sequences revealed that a Mu 1-like element was inserted 31 bp 5' from the transcriptional start site of Adh1-3F1124 gene. The insertion of the Mu element creates an additional TATA box by duplicating the 9 bp genomic sequence--ATATAAATC--at the site of insertion. Consequently, there are two potentially functional TATA sequences, separated by the 1.85 kbp Mu element, 5' to the transcriptional start site. It is not yet understood how such an arrangement alters the organ-specific expression of Adh1.

Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.

Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20% of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in SalI-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 +/- 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.

Characterization of a cloned repetitive DNA sequence concentrated on the human X chromosome.

A tandemly repeated DNA sequence organized predominantly, if not entirely, in a specific manner on the human X chromosome has been cloned in pBR322 and characterized. The sequence was detected as a 2-kilobase band in ethidium bromide-stained agarose gels of BamHI-digested total human nuclear DNA. Although in situ hybridization of the cloned sequence to human metaphase chromosomes showed a single major site of hybridization at the centromere region of the X chromosome and minor sites of hybridization at several autosomal centromeres, Southern blot analysis of restricted total human DNA indicated that the cloned probe is related to other repeated DNAs, particularly the human alphoid DNAs. Restriction enzyme analysis of the cloned fragment revealed an internal repeat structure based upon multiples of 170 base pairs, confirming this relatedness. All available data, however, suggest that the 2-kilobase spacing of BamHI sites within the repeat may be specific to the X chromosome.