Roy L. Fuchs

Assessment of the allergenic potential of foods derived from genetically engineered crop plants

This article provides a science-based, decision tree approach to assess the allergenic concerns associated with the introduction of gene products into new plant varieties. The assessment focuses on the source from which the transferred gene was derived. Sources fall into three general categories: common allergenic food proteins; less common allergenic foods or other known allergen sources; and sources with no history of allergenicity. Information concerning the amino acid sequence identity to known allergenic proteins, in vitro and/or in vivo immunologic assays, and assessment of key physiochemical properties are included in reaching a recommendation on whether food derived from the genetically modified plant variety should be labeled as to the source of the transferred gene. In the end, a balanced judgement of all the available data generated during allergenicity assessment will assure the safety of foods derived from genetically engineered crops. Using the approaches described here, new plant varieties generated by genetic modification should be introduced into the marketplace with the same confidence that new plant varieties developed by traditional breeding have been introduced for decades.

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready soybeans

Abstract We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready® soybeans. Western blotting and enzyme linked immunosorbent assay (ELISA) procedures were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the major processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup® herbicide. The western blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibody with biotinylated Protein-G. Detection of this complex is accomplished using horseradish-peroxidase (HRP) labeled NeutrAvidin™ and signal development by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been subjected to processing conditions typically employed by the food industry, thereby enabling development of an ELISA method. The ELISA for measurement of CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third biotin labeled monoclonal anti-rabbit antibody. Sandwich formation is detected using HRP labeled NeutrAvidin™ with color development using TMB substrate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was reduced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP4 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% Roundup Ready soybean mixed with conventional processed soybean fractions, thereby making the ELISA an acceptable method to assess CP4 EPSPS protein in processed soybean fractions. Data on sensitivity, accuracy, precision and specificity, established that the western blot and ELISA methods are appropriate for compliance with the EC Novel Foods Regulation.

Digestive Stability in the Context of Assessing the Potential Allergenicity of Food Proteins

Assessment of the potential allergenicity of proteins introduced into food crops through biotechnology is required by international regulatory agencies that govern the release and production of genetically modified plants. Currently, one aspect of this assessment includes analysis of the protein in a simulated gastric fluid (SGF) assay that tests the digestibility of the target protein to pepsin. The logic behind this test was that proteins that are nutritionally desirable tend to be rapidly digested and have greater bioavailability of amino acids than stable proteins. In addition, proteins that are highly digestible would be expected to have less opportunity to exert adverse health effects when consumed. The assay was not meant to predict the fate of the protein of interest under in vivo conditions, but rather to evaluate the susceptibility of the protein to digestion under fixed conditions in vivo. The purpose is to provide information that, in conjunction with other evidence, would be useful in predicting whether a dietary protein may become a food allergen. Therefore, the relationship of the resistance to digestion by pepsin and the likelihood that a dietary protein is an allergen was identified as a means of aiding the assessment of proteins added to commodity crops through biotechnology. In this article, we discuss the predictive value of this assay and the practical and theoretical aspects of allergen resistance to pepsin digestion in the context of food safety.

Preventing food allergy : Emerging technologies

Food allergy represents a largely avoidable threat. Technologies are presently available or are under development that better define the molecules (allergens) responsible for food allergy, better detect the presence of allergens, and can reduce or even eliminate specific allergens from the food supply. The incidence of food allergy and fatal anaphylaxis triggered by food allergens is expected to decline as a result of the application of a combination of key technologies such as biotechnology, bioinformatics and immunodiagnostics, and of the Internet, as well as following the development of well-defined science-based public health policies.

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

Safety Assessment of Insect-Protected Cotton

Publisher Summary This chapter provides information on the methods for developing Bollgard cotton event 531 and a summary of the food, feed, and environmental safety studies that support the safety of the Bollgard cotton. It discusses molecular characterization, the safety of the produced proteins, food/feed composition, and environment. On the basis of this evaluation, Bollgard cotton and its processed factions are found to be comparable to conveniently bred cotton, taking into consideration the natural variation seen among cotton varieties. Cotton is the leading plant fiber crop in the world, and the most important in the United States. Lepidopteran insects are the main pest problem on most cotton grown in the U.S. The incorporation of Bt protein into cotton reduces and in some cases eliminates the need to spray insecticides. The chapter discusses how Bollgard cotton is produced and the analyses that are performed to assess its nutritional and compositional equivalence to conventional varieties. Its molecular characterization is analyzed. Cry1Ac and NPTII protein levels in Bollgard cotton are discussed and their safety assessed. A compositional analysis and nutritional assessment of Bollgard cotton is provided, horizontal gene transfer and the assessment of marker genes are described, and an environmental assessment is provided.

Cloning and Expression of a Serratia Marcescens Gene Encoding Chitinase

Insects, fungi and nematodes contain chitin as integral structural components at one or more stages of their life cycles. Chitinase, either alone or in combination with other hydrolytic enzymes, exerts deleterious effects on representatives of each of these classes of organisms. Engineering plant specific rhizosphere or phylloplane colonizing bacteria to express and secrete chitinase may result in the production of biocontrol agents targeted at specific plant pathogens. A cosmid bank was constructed in the broad host range cosmid, pLAFRl, with partially EcoRI-restricted Serratia marcescens chromosomal DNA to clone one or more of the S. marcescens genes encoding chitinase. Four independent chitinase-positive clones were isolated, characterized and shown to share a common 9.5 kb EcoRI fragment apparently encoding the same 57 kd chitinase protein, the most abundant chitinase produced by S. marcescens. Chitinase is expressed in both Escherichia coli and Pseudomonas fluorescens at a low level in the original cosmid constructs and in constructs which contain the 9.5 kb EcoRI fragment cloned into a pKT230 derived plasmid. A 2.4 kb fragment generated from the 9.5 kb fragment by EcoRI “star” cleavage was inserted downstream of the Pbla promoter, resulting in a highly expressed chitinase construct in both E. coli and P. fluorescens.

Plant Biotechnology Products with Direct Consumer Benefits

Publisher Summary This chapter focuses on research efforts in biotechnology to genetically enhance crops with attributes, such as improved nutritional value and food quality, which more directly benefit customers. It summarizes the research reported in the scientific literature on potential new crop products with enhanced nutritional value or quality developed by biotechnology. Improved nutritional qualities in macronutrients, micronutrients—such as vitamins and minerals— other functional components, and special products—such as vaccines—are discussed. Macronutrients include fats, proteins, and carbohydrates. Phytochemicals or phytonutrients, including isoflavones, sterols, phytoestrogens, and anthocyanins are the functional components discussed. Special products include infant formula and human protein in plants. Products with enhanced quality traits are described. These involve enhancing freshness in fruits and vegetables, improving flavor and sweetness, preventing enzymatic browning, and improving functional characteristics using genetic enhancement. Reducing or eliminating allergens and other undesirable components is also detailed. The most common allergens exist in nuts, soy, peanuts, and wheat. Glucosinolates, steroidal glycoalkaloids, fungi, and caffeine content are discussed.