Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds (Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Expression of de novo high-lysine α-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants

We have designed protein molecules based on an -helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean -conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean -conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.     

Expression analysis of human β-secretase in transgenic tomato fruits

An emerging strategy in biomanufacturing involves using transgenic plants to express recombinant pharmaceutical and industrial proteins in large quantities. β-Site APP cleaving enzyme 1 (β-secretase 1, BACE1) is an enzyme involved in the abnormal production of Aβ42, the major component of senile plaques in Alzheimer's disease (AD). Thus, BACE1 represents a key target protein in the development of new potential drugs to treat Alzheimer's disease. We aimed to develop a tomato-derived recombinant BACE1 (rBACE1) protein to serve as a vaccine antigen that would promote an immune response. We utilized a plant expression cassette, pE8BACE, to optimize BACE1 expression in tomato fruits. Polyemerase chain reaction and Southern blot analyses verified integration of the BACE1 gene into the plant genome. Northern and Western blot analyses demonstrated successful mRNA and protein expression of rBACE1, respectively; the Sensizyme assay kit estimated the expression level of rBACE1 protein at 136 ± 7 ng mg?1 total soluble protein. The tomato-derived rBACE1 retains its activity for a long storage period at cool or room temperature, and is highly resistant to degradation in conditions such as low acidity. Tomato-derived rBACE1 was severely degraded by heat or boiling. The proteolytic activity of tomato-derived rBACE1, confirmed by fluorescence resonance transfer assay, was similar to that of a commercial sample of Escherichia coli-derived BACE1.     

Improvement of β-amylase thermostability in transgenic barley seeds and transgene stability in progeny

The genetic improvement of enzymes important in the brewing process is one of the main goals of barley biotechnology. For the improvement of -amylase thermostability in barley seeds, we have already constructed a mutant thermostable -amylase gene, using site-directed mutagenesis and random mutagenesis to achieve the substitution of seven amino acids of the original barley -amylase. This sevenfold-mutant barley -amylase showed a thermostability increased by 11.6 °C compared to the original enzyme. In the present article, a thermostable -amylase gene under the control of the barley -amylase promoter was introduced to barley protoplasts, and fertile plants were generated from 9 independent transgenic lines. Subsequent analyses indicated that the thermostable -amylase gene was expressed and -amylase activity derived from both native and modified genes was detected in the seeds of 6 transgenic lines. The transgene was stably transmitted to progeny, and thermostable -amylase was synthesized in T₄ seeds, demonstrating that our strategy is applicable for the improvement of seed quality for industrial utilization.     

Expression of the G-protein α-subunit gustducin in mammalian spermatozoa

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit α-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether α-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-α-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the α-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether α-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for α-gustducin. Although a possible function for α-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein α-subunit may process intracellular signals controlling sperm motility. Johanna Fehr and Dorke Meyer contributed equally to this work.     

Purification and some properties of γ-conglycinin in soybean seeds

A 7S globulin (γ-conglycinin) which was one of four major antigenic components in soybean globulins was purified and found to be homogeneous on ultracentrifugation, disc electrophoresis, immunoelectrophoresis and disc electrofocusing by gel filtration, preparative-scale disc electrophoresis and two kinds of affinity chromatography. Subsequently, some physico-chemical properties of the protein were determined. The sedimentation coefficient, isoelectric point, MW and diffusion constant were 6·55S, pH 5·80, 104000 and 5·80 × 10^?7 cm²/sec, respectively. The protein was a glycoprotein which contained 5·49% total carbohydrate per protein. The protein did not aggregate and dissociate with a change of ionic strength from 0·1 to 0·5.     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Characterization of transgenic rice plants over-expressing the stress-inducible β-glucanase gene Gns1

The Gns1 gene of rice (Oryza sativa L. japonica) encodes 1,3;1,4- glucanase (EC 3.2.1.73), which hydrolyzes 1,3;1,4--glucosidic linkages on 1,3;1,4--glucan, an important component of cell walls in the Poaceae family. RNA and protein gel blot analyses demonstrated that blast disease or dark treatment induced the expression of the Gns1 gene. To assess the function of the Gns1 gene in disease resistance, we characterized transgenic rice plants constitutively expressing the Gns1 gene. The introduced Gns1 gene was driven by the CaMV 35S promoter and its products were found in the apoplast and accumulated in up to 0.1% of total soluble protein in leaves. Although transgenic plants showed stunted growth and impaired root formation, fertility, germination, and coleoptile elongation appeared unaffected compared to non-transgenic control plants, indicating that Gns1 does not play a crucial role in rice germination and coleoptile elongation. When transgenic plants were inoculated with virulent blast fungus (Magnaporthe grisea), they developed many resistant-type lesions on the inoculated leaf accompanying earlier activation of defense-related genes PR-1 and PBZ1 than in control plants. Transgenic plants spontaneously produced brown specks, similar in appearance to those reported for an initiation type of disease-lesion-mimic mutants, on the third and fourth leaves and occasionally on older leaves without inoculation of pathogens. Expression of the two defense-related genes was drastically increased after the emergence of the lesion-mimic phenotype.     

Expression of fungal thermotolerant endo-1,4-β-glucanase in transgenic barley seeds during germination

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4--glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI -amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. A decrease in the soluble -glucan content in the wort improves the filtration rate of beer.     

Co-expression of α′ and β subunits of β-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins

A transgenic rice that produces both the α′ and β subunits of β-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the α′ subunit in the α′β-transgenic rice was slightly lower than that in the transgenic rice producing only the α′ subunit, the accumulation level of the β subunit in the α′β-transgenic rice was about 60% higher than that in the transgenic rice producing only the β subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the β subunit formed heterotrimers with the α′ subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the β subunit in the α′β-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the α′ and β subunits are localized in a low electron-dense region of protein body-II (PB-II) and that α′ homotrimers in the α′β-transgenic rice seeds seem to accumulate outside of this low electron-dense region.