4 resultados para Phage Purification

em Universidad Politécnica de Madrid


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The polysilicon market is experiencing tremendous changes due to the strong demand from Photovoltaics (PV), which has by far surpassed the demand from Microelectronics. The need of solar silicon has induced a large increase in capacity, which has now given a scenario of oversupply, reducing the polysilicon price to levels that put a strong pressure on the cost structure of the producers. The paper reports on the R&D efforts carried out in the field of solar silicon purification via the chlorosilane route by a private-public consortium that is building a pilot plant of 50-100 tonnes/year, that will synthesize trichlorosilane, purify it and deposit ultrapure silicon in an industrial-size Siemens type reactor. It has also capabilities for ingot growth and material characterization. A couple of examples of the progress so far are given, the first one related to the recycling scheme of chlorinated compounds, and the second to the minimization of radiation losses in the CVD deposition process, which account for a relevant part of the total energy consumption. In summary, the paper gives details on the technology being developed in our pilot plant, which offers a unique platform for field-testing of innovative approaches that can lead to a cost reduction of solar silicon produced via the chlorosilane route.

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In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

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Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL?1 and 100 ng mL?1 of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g?1 (110e120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.