An integral part of the safety assessment of genetically modified plants is consideration of possible human health effects, especially food allergy. Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult because of the absence of valid methods and models. Food allergens may share physicochemical properties that distinguish them from nonallergens, properties that may be used as a tool to predict the inherent allergenicity of proteins newly introduced into the food supply by genetic engineering. One candidate property is stability to digestion. We have systematically evaluated the stability of food allergens that are active via the gastrointestinal tract in a simple model of gastric digestion, emphasizing the major allergens of plant-derived foods such as legumes (peanuts and soybean). Important food allergens were stable to digestion in the gastric model (simulated gastric fluid). For example, soybean beta-conglycinin was stable for 60 min. In contrast, nonallergenic food proteins, such as spinach ribulose bis-phosphate carboxylase/oxygenase, were digested in simulated gastric fluid within 15 sec. The data are consistent with the hypothesis that food allergens must exhibit sufficient gastric stability to reach the intestinal mucosa where absorption and sensitization (development of atopy) can occur. Thus, the stability to digestion is a significant and valid parameter that distinguishes food allergens from nonallergens.

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Increased expression of the insect control protein genes ofBacilus thunrngiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modIfied (3% nucleotide difference) and to fully modified (21% nucleotide difference) crylA(b) and crylA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified crylA(b) gene had a 10-fold higher level ofinsect control protein and plants with the fully modified crylA(b) had a 100-fold higher level of CryIA Insect control proteins from a prokaryotic source, Bacillus thuringiensis var. kurstaki (B.t.k.; ref. 1) are specific for lepidopteran insects and exhibit no activity against humans, other vertebrates, and beneficial insects (2). These properties have made the genes of these insect-specific proteins attractive candidates for genetic modification of crops for protection against lepidopteran pests. Genes encoding lepidopteran-specific insect control proteins have been cloned and sequenced. Truncated genes, which produce insecticidally active protein, have been expressed in tomato (3), tobacco (4), and cotton (5). Field tests of these plants revealed that higher levels of insect control protein in the plant tissue would be required to obtain commercially useful plants (6).The insect control proteins are highly expressed in their natural host, B. thuringiensis. Up to 50% of the total protein in sporulated cultures ofB.t.k. are the insect control proteins deposited as crystals within the cell. Insect control protein genes are expressed well in Escherichia coli (7) or Pseudomonas (8). Poor expression in plants is a well-reported characteristic of the B.t.k. insect control proteins. Truncating the gene, keeping essentially the N-terminal half of the protein intact, results in improved expression of the gene in plants to barely detectable levels (3, 4). The use ofdifferent promoters, fusion proteins, and leader sequences has not significantly increased insect control protein gene expression (4, 9).We hypothesized that a gene with a sequence adapted for a Gram-positive prokaryote may not be the appropriate coding sequence for efficient plant expression. Examination of the insect control protein gene coding sequence indicated that it differs significantly from plant genes in G+C content. Multiple sequences motifs that are not common in the coding region of plant genes were found to be common in the wild-type (WT) crylA(b) sequence. These included localized regions of A+T richness resembling plant introns (10), potential plant polyadenylylation signal sequences (11), ATTTA sequences, which have been shown to destabilize mRNA in other systems (12), and codons rarely used in plants...

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We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.