Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.     

Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements

Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 mug of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 mug/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 mug of cadmium per g or 500 mug of copper per g of germ.     

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.     

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.     

Purification of an acidic recombinant protein from transgenic tobacco

Tobacco has proven to be a promising alternative for the production of recombinant therapeutic proteins and offers numerous advantages over other plants as a host system. However, the recovery and purification steps needed to obtain a protein at high recovery and purity have not been well investigated. In this study, a process was developed to purify a model acidic protein, recombinant beta-glucuronidase (rGUS) from transgenic tobacco leaf tissue, in three main steps after extraction: polyelectrolyte precipitation, hydrophobic interaction chromatography (HIC), and hydroxyapatite chromatography (HAC). Using this three-step process, up to 40% of the initial rGUS activity could be recovered to near homogeneity as judged by SDS-PAGE. This work demonstrates that acidic recombinant proteins expressed in tobacco may be purified to high yield with high purity in a minimal amount of steps that are suitable for scale-up. Furthermore, the general steps used in this process may suggest that a wide variety of acidic recombinant proteins may be purified in a similar manner from transgenic tobacco or other leafy crops.     

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants     

Improved recovery of active recombinant laccase from maize seed

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.     

黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究

GNK2-1为一种来自银杏(Ginkgo biloba)种仁的新型抗真菌蛋白, 具有较强的真菌抗性且性质稳定。序列分析表明,其结构与所有已知的抗真菌蛋白不同, 而与富含半胱氨酸的植物类受体激酶的胞外结构域相似。为探索GNK2-1基因在黄瓜(Cucumis sativus)抗病反应中的作用, 利用基因重组技术构建了GNK2-1的高效组成型表达载体, 并利用根癌农杆菌(Agrobacterium tumefaciens)介导转入黄瓜栽培品种农城3号(Cucumis sativus ‘Nongcheng No.3’)基因组中。通过对获得的抗性植株进行PCR、RT-PCR和Western blot检测分析, 结果表明GNK2-1基因可在T₀代转基因植株中转录表达, 并能在T₁代转基因黄瓜中稳定遗传。离体枯萎病抗性鉴定结果表明, 转GNK2-1基因的黄瓜对枯萎病的抗性增强, GNK2-1可以作为黄瓜抗病性改良的潜在基因资源。     

黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究

GNK2-1为一种来自银杏（Ginkgo biloba）种仁的新型抗真菌蛋白,具有较强的真菌抗性且性质稳定。序列分析表明,其结构与所有已知的抗真菌蛋白不同,而与富含半胱氨酸的植物类受体激酶的胞外结构域相似。为探索GNK2-1基因在黄瓜（Cucumis sativus）抗病反应中的作用,利用基因重组技术构建了GNK2-1的高效组成型表达载体,并利用根癌农杆菌（Agrobacterium tumefaciens）介导转入黄瓜栽培品种农城3号（Cucumis sativus＇ Nongcheng No.3＇）基因组中。通过对获得的抗性植株进行PCR、RT-PCR和Western blot检测分析,结果表明GNK2-1基因可在T0代转基因植株中转录表达,并能在T1代转基因黄瓜中稳定遗传。离体枯萎病抗性鉴定结果表明,转GNK2-1基因的黄瓜对枯萎病的抗性增强,GNK2-1可以作为黄瓜抗病性改良的潜在基因资源。     

Autohydrolysis of plant polysaccharides using transgenic hyperthermophilic enzymes

Commercial bioprocessing of plant carbohydrates, such as starch or cellulose, necessitates the use of commodity enzyme additives to accelerate polysaccharide hydrolysis. To simplify this procedure, transgenic plant tissues constitutively producing commodity enzymes were examined as a strategy for accelerating carbohydrate bioprocessing. Hyperthermophilic glycosyl hydrolases were selected to circumvent enzyme toxicity, because such enzymes are inactive at plant growth temperatures and are therefore physiologically benign. Transgenic tobacco lines were established that produced either a hyperthermophilic alpha-glucosidase or a beta-glycosidase using genes derived from the archaeon Sulfolobus solfataricus. Western blot and immunoprecipitation analyses were used to demonstrate the presence of recombinant enzymes in plant tissues. Transgenic enzyme levels exhibited an unusual delayed pattern of accumulation while their activities survived plant tissue preservation. Transgenic plant protein extracts released glucose from purified polysaccharide substrates at appreciable rates during incubation in high-temperature reactions. Glucose was also produced following enzymatic treatment of plant extracts enriched for endogenous polysaccharides. Direct conversion of plant tissue into free sugar was evident using whole plant extracts of either transgenic line, and could be significantly accelerated in a synergistic manner by combining transgenic line extracts.     

Biological Activity of Human Granulocyte-Macrophage Colony Stimulating Factor is Maintained in a Fusion with Seed Glutelin Peptide

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.     

Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration

Summary Transgenic Phaseolus vulgaris or common bean has been produced using electric-discharge particle acceleration. The method uses particle acceleration to introduce DNA into bean seed meristems. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 0.03% germline transformed plants/shoot. We have been able to recover transgenic plants using both GUS and herbicide screening to introduce the gus, bar, and bean golden mosaic virus coat protein genes into the navy bean cultivar, Seafarer. The transgenic plants have been characterized over 5 generations of self-fertilization with no loss of introduced genes or expression. In addition, several families have been crossed with non-transgenic parents and these plants also show expected inheritance patterns. The introduced bar gene has been shown to confer strong resistance in transgenic beans to basta herbicide application in the greenhouse.Abbreviations BGMV bean golden mosaic virus - PAT phosphinothricin acetyltransferase     

Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Summary Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance. The University of Wisconsin and the USDA neither guarantee nor warrant the standard of the products named herein, and the use of the name by University of Wisconsin or USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.     

Commercial production of avidin from transgenic maize: characterization of transformant,production, processing,extraction and purification

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces.An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at –29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

Aminopeptidase activity associated with alpha1-conarachin (peanut protein)

Aminopeptidases were investigated in protein extracts of dormant and germinated peanut cotyledons by electrophoretic immunochemical techniques. Considerable activity was observed in protein zones that migrated toward the anode after simple electrophoresis. Of the immunogenic proteins in dormant seed, aminopeptidase activity was associated only with the immunocomplex of α₁-conarachin, a globular protein in peanuts. The specific aminopeptidase activity of total extracts was marginally higher than that of purified α-conarachin. Specific iso-enzyme(s) adsorption on the antigen-antibody complex of α₁-conarachin might be attributed in part, to these phenomena. Reactions of protein extracts from germinated cotyledons with immune sera made against protein in germinated tissues—roots and cotyledons—showed the identical associative interaction. Some of the determinant groups on α₁-conarachin in the germ and cotyledon were apparently maintained during early phases of germination.     

High Lysine and High Tryptophan Transgenic Maize Resulting from the Reduction of Both 19- and 22-kD α-zeins

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD α-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, asparate and glutamate, was observed in the zein reduction kernels.     

GmTMT2a from soybean elevates the α-tocopherol content in corn and Arabidopsis

Tocochromanol, or vitamin E, plays a crucial role in human and animal nutrition and is synthesized only by photosynthetic organisms. γ-Tocopherol methyltransferase (γ-TMT), one of the key enzymes in the tocopherol biosynthetic pathway in plants, converts γ, δ-tocopherols into α-, β-tocopherols. Tocopherol content was investigated in 15 soybean cultivars and GmTMT2 was isolated from five varieties based on tocopherol content. GmTMT2a was expressed in E. coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. Overexpression of GmTMT2a enhanced α-tocopherol content 4–6-fold in transgenic Arabidopsis, and α-tocopherol content increased 3–4.5-fold in transgenic maize seed, which correlated with the accumulation of GmTMT2a. Transgenic corn that is α-tocopherol-rich may be beneficial for animal health and growth.     

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.