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1.
Methods Mol Biol ; 483: 209-21, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19183901

RESUMO

Over the last decade, plant-based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated in a range of plant crops. Although the commercial exploitation is still pending, today various plant-based expression technologies have reached significant milestones through clinical testing in humans. Each of the protein manufacturing platforms in plants has specific benefits and drawbacks. We have engaged in comparing some of these production systems with respect to their performance: protein yield and quality. Using a specific tester protein (aprotinin), it was shown that functional aprotinin can be manufactured in plants in substantial amounts, as illustrated in this chapter.


Assuntos
Aprotinina/biossíntese , Nicotiana/genética , Preparações Farmacêuticas , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Aprotinina/química , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transgenes
2.
Plant Biotechnol J ; 6(3): 309-20, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18266824

RESUMO

Aprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.


Assuntos
Aprotinina/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Nicotiana/citologia , Nicotiana/genética , Inibidores de Proteases/metabolismo , Sequência de Aminoácidos , Aprotinina/genética , Regulação da Expressão Gênica de Plantas/genética , Engenharia Genética , Folhas de Planta/citologia , Plantas Geneticamente Modificadas , Transporte Proteico , Proteínas Recombinantes , Tilacoides
3.
Plant Biotechnol J ; 6(1): 46-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17944820

RESUMO

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.


Assuntos
Cloroplastos/metabolismo , Dissulfetos/metabolismo , Nicotiana/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos , Plantas Geneticamente Modificadas/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/genética , Nicotiana/genética , Transformação Genética
4.
Plant Biotechnol J ; 5(1): 118-33, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17207262

RESUMO

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.


Assuntos
4-Hidroxifenilpiruvato Dioxigenase/genética , Glycine max/genética , Herbicidas/toxicidade , Nicotiana/genética , Plastídeos/genética , Pseudomonas fluorescens/genética , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tolerância a Medicamentos/genética , Pseudomonas fluorescens/enzimologia , Proteínas Recombinantes/metabolismo , Nicotiana/efeitos dos fármacos
5.
Methods Mol Biol ; 1132: 345-54, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24599865

RESUMO

The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology.


Assuntos
Biolística/métodos , Cloroplastos/genética , Glycine max/genética , Antibacterianos/farmacologia , DNA Ribossômico/genética , Resistência a Medicamentos/genética , Nucleotidiltransferases/genética , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , RNA Ribossômico 16S/genética , Sementes/genética , Espectinomicina/farmacologia , Transformação Genética , Transgenes/genética
6.
Plant Physiol ; 150(3): 1474-81, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19458113

RESUMO

Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP). In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco. Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and limiting pleiotropic effects.


Assuntos
Adaptação Fisiológica , Cloroplastos/metabolismo , Nicotiana/genética , Proteínas Recombinantes/metabolismo , Aminoácidos/metabolismo , Cloroplastos/genética , Vetores Genéticos , Dados de Sequência Molecular , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismo
7.
Transgenic Res ; 15(3): 305-11, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16779646

RESUMO

The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.


Assuntos
Técnicas Genéticas , Glycine max/genética , Plantas Geneticamente Modificadas , Plastídeos/genética , Proteínas Recombinantes/química , Ágar/química , DNA/metabolismo , DNA Intergênico/genética , Resistência a Medicamentos , Vetores Genéticos , Genótipo , Modelos Genéticos , Fenótipo , Plastídeos/metabolismo , Espectinomicina/farmacologia , Fatores de Tempo
8.
Plant Physiol ; 134(1): 92-100, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14684842

RESUMO

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.


Assuntos
Nicotiana/genética , Nicotiana/metabolismo , Ácido Chiquímico/metabolismo , Tocotrienóis/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , 4-Hidroxifenilpiruvato Dioxigenase/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , DNA Recombinante/genética , Resistência a Medicamentos/genética , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Genes Fúngicos , Genes de Plantas , Engenharia Genética , Herbicidas/farmacologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Prefenato Desidrogenase/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Vitamina E/metabolismo
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