RESUMEN
The speed of simulated gastric digestion of proteins expressed in genetically engineered (GE) crops is commonly used to inform the allergenicity risk assessment. However, persistence of purified proteins in simulated gastric fluid (SGF) is poorly correlated with the allergenic status of proteins. It has been proposed that the plant or food matrix may affect the digestion of proteins and should be considered in interpreting digestion results. Here the SGF digestion of several GE proteins both as purified preparations and in soybean, corn, and cotton seed/grain extracts (in-matrix) are compared. Cry1F, Cry1Ac, phosphinothricin acetyltransferase (PAT), aryloxyalkanoate dioxygenase-1 (AAD-1), aryloxyalkanoate dioxygenase-12 (AAD-12), and double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) were all found to rapidly digest both as purified protein preparations and in seed/grain extracts from GE crops expressing these proteins. Based on these results, purified protein from microbial sources is a suitable surrogate for proteins in-matrix when conducting SGF digestion studies.
Asunto(s)
Alérgenos/metabolismo , Productos Agrícolas/metabolismo , Digestión , Alimentos Modificados Genéticamente , Jugo Gástrico/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Semillas/metabolismo , Alérgenos/efectos adversos , Alérgenos/genética , Alérgenos/inmunología , Productos Agrícolas/efectos adversos , Productos Agrícolas/genética , Productos Agrícolas/inmunología , Hipersensibilidad a los Alimentos/etiología , Hipersensibilidad a los Alimentos/inmunología , Inocuidad de los Alimentos , Alimentos Modificados Genéticamente/efectos adversos , Jugo Gástrico/química , Humanos , Proteínas de Plantas/efectos adversos , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Plantas Modificadas Genéticamente/efectos adversos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Proteolisis , Medición de Riesgo , Semillas/efectos adversos , Semillas/genética , Semillas/inmunología , Factores de TiempoRESUMEN
Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium Delftia acidovorans MC1 and is a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. (1) The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in Pseudomonas fluorescens (Pf) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and in vitro activity analyses demonstrated to be equivalent to the Pf-generated enzyme. Furthermore, results from in vitro kinetic analyses using a variety of plant endogenous compounds revealed activity with trans-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (kcat/Km) of AAD-12 using trans-cinnamate (51.5 M(-1) s(-1)) and IAA (8.2 M(-1) s(-1)) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dioxigenasas/química , Dioxigenasas/metabolismo , Glycine max/metabolismo , Herbicidas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Pseudomonas fluorescens/genética , Proteínas Bacterianas/genética , Dioxigenasas/genética , Expresión Génica , Resistencia a los Herbicidas , Herbicidas/farmacología , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Pseudomonas fluorescens/química , Pseudomonas fluorescens/metabolismo , Glycine max/química , Glycine max/efectos de los fármacos , Glycine max/genética , Especificidad por SustratoRESUMEN
A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.
Asunto(s)
Productos Agrícolas/enzimología , Aceites de Plantas/química , Semillas/química , Estearoil-CoA Desaturasa/metabolismo , Baculoviridae , Membrana Celular , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Valor Nutritivo , Plantas Modificadas Genéticamente , Estearoil-CoA Desaturasa/genéticaRESUMEN
To evaluate the potential of Bacillus thuringiensis (Bt) Cry1F protein accumulation in soil, transgenic corn containing event DAS-01507-1 encoding the cry1F gene was grown in three field sites for 3 consecutive yr, and the corn plants were incorporated into the soil through postseason tillage or no tillage each year. Soil samples were collected from these fields, and the level of Cry1F protein in these samples was determined using an enzyme-linked immunosorbent assay (ELISA) with a synthetic invertebrate gut fluid as an extraction buffer. The ELISA was validated in soil matrices over the concentration range of 18-180 ng/g dry weight, with a limit of detection of 4.5 ng/g dry weight. The assay was shown to have good accuracy and precision. No detectable Cry1F protein was found in any of the soil samples collected from the Cry1F corn fields. Soil also was bioassayed, and no biological activity was observed against Heliothis virescens neonates. These results indicate that the level of Cry1F protein accumulated in soil after 3-yr continuous planting of transgenic Cry1F corn is negligible.
Asunto(s)
Proteínas Bacterianas/análisis , Toxinas Bacterianas/análisis , Endotoxinas/análisis , Proteínas Hemolisinas/análisis , Plantas Modificadas Genéticamente/metabolismo , Suelo/análisis , Zea mays/metabolismo , Animales , Anticuerpos/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Bioensayo , Peso Corporal , Endotoxinas/genética , Ensayo de Inmunoadsorción Enzimática , Proteínas Hemolisinas/genética , Mariposas Nocturnas/efectos de los fármacos , Control Biológico de Vectores/métodos , Raíces de Plantas/metabolismo , Reproducibilidad de los Resultados , Medición de Riesgo/métodos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Agricultural biotechnology companies have been asked to provide intact transgenic seed to regulatory agencies as reference materials for evaluating transgene and protein detection methods (PCR and immunoassay). Due to intellectual-property and product-stewardship considerations, submission of devitalized seed prior to regulatory approval is preferable in any given country. Commonly used devitalization procedures, such as heating or autoclaving, degrade the protein and/or DNA rendering the seed unfit as a reference material for these tests. A novel method for devitalizing seed was developed that involves hydration, freezing in liquid nitrogen, and lyophilization. The devitalization method described here was found to preserve the transgenic DNA and protein in cotton (Gossypium hirsutum) and maize (Zea mays) seed allowing its use as a reference material for evaluating detection methods.
Asunto(s)
ADN de Plantas/química , ADN de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/genética , Semillas/química , Semillas/genética , Biotecnología , Southern Blotting , Western Blotting , Criopreservación , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Liofilización , Ingeniería Genética/legislación & jurisprudencia , Ingeniería Genética/normas , Germinación , Gossypium/química , Gossypium/genética , Preservación Biológica/métodos , Proteínas Recombinantes/genética , Estándares de Referencia , Semillas/crecimiento & desarrollo , Zea mays/química , Zea mays/genéticaRESUMEN
A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.
Asunto(s)
Proteínas Bacterianas/análisis , Toxinas Bacterianas/análisis , Endotoxinas/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Gossypium/química , Proteínas Hemolisinas/análisis , Insecticidas/análisis , Plantas Modificadas Genéticamente/química , Anticuerpos Monoclonales , Toxinas de Bacillus thuringiensis , Sensibilidad y EspecificidadRESUMEN
Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.