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1.
J Agric Food Chem ; 69(35): 10358-10370, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428040

RESUMO

The advancement of mass spectrometry provides advantages for transgenic protein characterization in support of safety assessments of genetically modified crops. Here, we describe how matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) in combination with intact mass and bottom-up analyses can be applied to achieve high confidence in the sequences of transgenic proteins expressed in plants and establish the biochemical equivalence of microbially produced protein surrogates. ISD confirmed 40-60 near terminal residues regardless of the protein size, including the improvement of the coverage of cysteine-rich proteins by the reduction/alkylation of disulfide bonds. Negative ISD significantly improved spectral quality and sequence coverage of acidic proteins. Various post-translational modifications, such as terminal truncations and N-terminal methionine excision and acetylation, were identified in plant-produced proteins by top-down MS. Finally, we demonstrated that a combination of top-down and bottom-up analyses provides high confidence in sequence equivalence of plant and microbially produced proteins.


Assuntos
Produtos Agrícolas , Proteínas , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Transgenic Res ; 29(1): 109-124, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31781961

RESUMO

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.


Assuntos
Toxinas de Bacillus thuringiensis/administração & dosagem , Bacillus thuringiensis/metabolismo , Besouros/efeitos dos fármacos , Endotoxinas/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Plantas Geneticamente Modificadas/metabolismo , Zea mays/metabolismo , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Endotoxinas/metabolismo , Glicosilação , Proteínas Hemolisinas/metabolismo , Plantas Geneticamente Modificadas/genética , Zea mays/genética
3.
Clin Transl Allergy ; 8: 30, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30116520

RESUMO

Background: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

4.
Environ Entomol ; 47(2): 484-497, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29432611

RESUMO

Many studies have been conducted to investigate synergism among insecticidal proteins; however, a consensus on minimal data requirements and interpretation is lacking. While some have concluded that all additive predictive-type models should be abandoned, we advocate that additivity models can remain useful as assessment tools and that an appropriately designed interaction study will never systematically underestimate the existence of synergism, irrespective of which additivity model (or none at all) may be used. To generate the most meaningful synergy assessment datasets in support of safety assessments, we highlight two beneficial steps to follow: (i) select a testing model which is the most consistent with current knowledge regarding the action of the insecticidal proteins and (ii) avoid using bioassay methods which may result in excess response heterogeneity. We also outline other experimental design elements to consider, which may be crucial for conducting future studies of this type. A contrast of underlying testing assumptions associated with the additivity models is provided, along with a comprehensive review of interaction data for Cry1, Cry2, Cry3, Cry9, and Vip3A insecticidal proteins. Our review captures four recurrent findings: i) experiments reporting synergistic interactions are a minority, ii) the degree of synergism reported is low in magnitude, iii) reported interactions are sometimes equivocal/inconclusive due to unconfirmed model assumptions or other bioassay challenges, and iv) due to biological response variation many of the reported interactions may be artefactual. A brief overview of the positioning of interaction testing data in safety assessments of GM food crops is also provided.


Assuntos
Proteínas de Bactérias , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endotoxinas , Proteínas Hemolisinas , Insetos , Animais , Toxinas de Bacillus thuringiensis , Bioensaio , Modelos Teóricos
5.
J Insect Sci ; 17(2)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28355479

RESUMO

A shift toward transgenic crops which produce combinations of insecticidal proteins has increased the interest (Syngenta Seeds, Inc., Minnetonka, MN) in studying the potential for interactions amongst those proteins. We present a general testing method which accommodates proteins with nonoverlapping spectrums of activity. Our sequential testing approach first investigates groups of the proteins with overlapping activity; e.g., proteins active against Lepidoptera or Coleoptera, respectively. The Colby method is used to test for interactions within each respective group. Subsequently, the mixture of proteins within each group is regarded as a single entity and tests for interactions between the groups (when combined) is conducted using analysis of variance. We illustrate the method using Cry1Ab, Vip3Aa20, and Cry1F (a mixture of proteins active against Lepidoptera), and mCry3A and eCry3.1Ab (a mixture of proteins active against Coleoptera). These insecticidal proteins are produced by Bt11 × MIR162 × TC1507 × MIR604 × 5307 maize. We detected no interactions between Cry1Ab, Vip3Aa20, and Cry1F in tests using larvae of two different lepidopteran species, and possible slight antagonism between mCry3A and eCry3.1Ab with a coleopteran test species. We detected no effect of (eCry3.1Ab + mCry3A) on the potency of (Cry1Ab + Vip3Aa20 + Cry1F) to lepidopteran larvae, and no effect of (Cry1Ab + Vip3Aa20 + Cry1F) on the potency of (mCry3A + eCry3.1Ab) to coleopteran larvae. We discuss implications of these results for characterization of Bt11 × MIR162 × TC1507 × MIR604 × 5307 maize, and the value of the method for characterizing other transgenic crops that produce several insecticidal proteins.


Assuntos
Proteínas de Bactérias , Agentes de Controle Biológico , Besouros , Inseticidas , Lepidópteros , Animais , Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Bioensaio , Agentes de Controle Biológico/metabolismo , Besouros/metabolismo , Resistência a Inseticidas , Inseticidas/metabolismo , Larva/metabolismo , Lepidópteros/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas , Zea mays/genética , Zea mays/metabolismo
6.
Methods Mol Biol ; 1385: 259-70, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26614295

RESUMO

Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet.


Assuntos
Toxinas Bacterianas/genética , Bioensaio/métodos , Lepidópteros/efeitos dos fármacos , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Plantas/genética , Animais , Toxinas Bacterianas/farmacologia , Larva/efeitos dos fármacos , Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transgenes
7.
Regul Toxicol Pharmacol ; 69(2): 154-70, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24662477

RESUMO

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.


Assuntos
Produtos Agrícolas/genética , Alimentos Geneticamente Modificados , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Segurança , Ração Animal , Animais , Qualidade de Produtos para o Consumidor , Medição de Risco
8.
Transgenic Res ; 22(2): 445-60, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23065372

RESUMO

Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.


Assuntos
Escherichia coli/genética , Proteínas de Plantas/genética , Proteínas de Plantas/toxicidade , Plantas Geneticamente Modificadas/toxicidade , Animais , Produtos Agrícolas/genética , Produtos Agrícolas/toxicidade , Humanos , Camundongos , Proteínas de Plantas/efeitos adversos , Plantas Geneticamente Modificadas/efeitos adversos , Plantas Geneticamente Modificadas/genética , Medição de Risco
9.
J Agric Food Chem ; 53(4): 1231-6, 2005 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15713046

RESUMO

Flavonoids represent a large and important group of plant natural products that are ubiquitous in the plant kingdom. Epidemiological studies have shown the health benefits of a diet high in flavonoids. However, the dietary intake of flavonoids in most western populations is limited, creating a need to find alternative food sources for these polyphenolic secondary metabolites. The domestication of many of our cultivated food crops has resulted in alterations in the biosynthetic pathways of many essential micronutrients and vitamins through inadvertent counterselection against nutritional traits in favor of agronomic ones. Flavonoids are nearly absent from fruits of cultivated tomato (Lycopersicon esculentum Mill.), a major vegetable in human diets. Previous attempts to restore the flavonoid pathway in tomato fruits have been limited to transgenic strategies, suggesting that the problem was intractable through traditional methods. Here, we describe for the first time a nontransgenic metabolic engineering approach to developing a high flavonoid tomato using a wild tomato species (Lycopersicon pennelliiv. puberulum) and demonstrate the opportunities for restoring functional pathways using the genetic resources of wild species, resulting in production of healthier foods.


Assuntos
Flavonoides/análise , Frutas/química , Lycopersicon esculentum/química , Lycopersicon esculentum/genética , Cruzamento , Flavonoides/biossíntese , Flavonoides/genética , Expressão Gênica , Engenharia Genética , Hibridização Genética
10.
Phytochemistry ; 65(1): 31-41, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14697269

RESUMO

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.


Assuntos
Flavonoides/metabolismo , Glucosiltransferases/metabolismo , Metiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Clonagem Molecular , Sequência Consenso , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Flavonoides/química , Glucosiltransferases/química , Glucosiltransferases/genética , Mentha piperita/enzimologia , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Cebolas/enzimologia , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato
11.
Phytochemistry ; 64(6): 1069-76, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14568073

RESUMO

Two UDP-glucose-dependent flavonoid glucosyltransferases (EC 2.4.1.-) isolated from the epidermal layer of yellow onion (Allium cepa) were functionally expressed in Escherichia coli and their substrate specificity investigated. The two enzymes exhibited different substrate- and regio-specificity profiles. A. cepa UGT73G1 used a wide range of different flavonoid substrates including flavonoids not naturally occurring in onion. Regiospecificity was indicated for hydroxyl-groups of the C-3, C-7 and C-4' positions of the flavan backbone structure to yield flavonoid mono- and diglucosides. In contrast, A. cepa UGT73J1 showed activity only with the flavonoid mono-glucoside isoquercitrin and the isoflavone aglycone genistein, with regiospecificity for the C-7 position. The regiospecificity for both enzymes included positions that are glucosylated in flavonoids of onion bulbs, indicating their involvement in flavonoid biosynthesis in A. cepa.


Assuntos
Flavonoides/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Cebolas/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Flavonoides/química , Glucosiltransferases/química , Glicosilação , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato
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