New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called “hard-to-treat” tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treats organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1–4 h, both β-gal:RTB and RTB:β-gal were processed to the ~64 kDa “activated” β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48 h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis – delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

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.

Mating by cellular fusion is essential for segregational and recombinational analysis of nuclear or cytoplasmic inheritance in Saccharomyces cerevisiae. However, a detailed description of the mating reaction at the molecular level has not been established. In part, this reflects the inadequacy of current experimental methods for studying mating in yeast. In heterothallic yeasts mating occurs when cells with complementary mating types are mixed under appropriate conditions. The conjugation reaction (1, 2) then progresses as a sequence of ordered events that can be subdivided on a gross visual and cytological basis into three principal phases: (a) mating initiation, which involves conjugant pairing and sexual agglutination, (b) cell fusion (plasmogamy), and (c) nuclear fusion (karyogamy). The completed reaction sequence yields a morphologically distinct zygote that can, depending on nutritional conditions, either proliferate mitotically or enter into meiotic development and ascospore formation.A prerequisite for studies aimed at understanding mating and sexuality is the ability to obtain sizable, homogeneous cell populations that undergo synchronous mating under defined conditions. We report here our initial studies on mating and a protocol for producing highly efficient synchronously mating populations of yeast in liquid minimal medium. From these populations, purified zygote suspensions are readily isolated in quantities suitable for extensive genetic and biochemical analyses. MATERIALS AND METHODSStrains. Two heterothallic haploid strains of S. cerevisiae containing complementary leucine and tryptophan markers were isolated as single spore clones: 5032A a leu2-27 tryl-1 and 5032B a leul-12 trp4. Growth Mating Procedure. The single cell fraction obtained from the zonal rotor was centrifuged to remove the sorbitol, resuspended in 100 ml of YNB to a density of 6 to 10 X 106 cells per ml in a 500-ml Erlenmyer flask, and shaken at 300. The beginning of incubation is defined as zero time for the mating reaction. A cell sample was immediately withdrawn from the mating mixture to determine the ratio of a and a unbudded cells obtained from the rotor. Samples taken throughout the first 180 min of the mating reaction were fixed immediately with an equal volume of 3.7% formaldehyde in 0.05 M phosphate buffer (pH 7.0).Zygote Isolation. After 120 and 150 min of incubation, 30-40 ml of the cell suspension was harvested by centrifugation, resuspended in l ml of water, sonicated for 5 sec, and then layered onto a 25-ml linear 10-30% sorbitol gradient. The gradient was centrifuged at 1000 rpm in a Sorvall HB4

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Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.