Andre Silvanovich

Bioinformatic Methods for Allergenicity Assessment Using a Comprehensive Allergen Database

Background: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. Methods: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. Results: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared ≧35% identity in an overlap of ≧80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. Conclusions: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

The Use of Next Generation Sequencing and Junction Sequence Analysis Bioinformatics to Achieve Molecular Characterization of Crops Improved Through Modern Biotechnology

The assessment of genetically modified (GM) crops for regulatory approval currently requires a detailed molecular characterization of the DNA sequence and integrity of the transgene locus. In addition, molecular characterization is a critical component of event selection and advancement during product development. Typically, molecular characterization has relied on Southern blot analysis to establish locus and copy number along with targeted sequencing of polymerase chain reaction products spanning any inserted DNA to complete the characterization process. Here we describe the use of next generation (NexGen) sequencing and junction sequence analysis bioinformatics in a new method for achieving full molecular characterization of a GM event without the need for Southern blot analysis. In this study, we examine a typical GM soybean [Glycine max (L.) Merr.] line and demonstrate that this new method provides molecular characterization equivalent to the current Southern blot‐based method. We also examine an event containing in vivo DNA rearrangement of multiple transfer DNA inserts to demonstrate that the new method is effective at identifying complex cases. Next generation sequencing and bioinformatics offers certain advantages over current approaches, most notably the simplicity, efficiency, and consistency of the method, and provides a viable alternative for efficiently and robustly achieving molecular characterization of GM crops.

The use of E-scores to determine the quality of protein alignments

In 2001, the FAO/WHO suggested a procedure for performing FASTA or BLAST searches, and a threshold of greater than 35% identity in 80 or greater amino acids to identify potential allergenic cross-reactivity of transgene encoded proteins in genetically enhanced crops. Transgene encoded proteins meeting or exceeding this threshold would require additional in vitro evaluation for allergy safety. In work described herein, a method to calculate an E-score threshold is proposed for utilizing the full capability of bioinformatics to accurately identify potential cross-reactive allergens. The threshold E-score, 3.9E-07, was produced using a test dataset of 7695 corn protein sequences and a method that entailed FASTA searches of the FARRP 7 allergen database with each of the dataset sequences using a conventional full length and an 80 amino acid sliding window FASTA comparison followed by an evaluation of E-score distribution. The results show that this E-score threshold identifies known corn allergens and it displays a false positive rate for known allergens that is comparable to that obtained from the 2001 FAO/WHO guidance. Furthermore, the E-score threshold is of sufficient stringency that it rejects the majority of false positive, composition-based anomalies and is 100% effective at identifying Bet v 1 cross-reactive allergens.

Bioinformatic Methods for Identifying Known or Potential Allergens in the Safety Assessment of Genetically Modified Crops

Agricultural crops have been genetically improved through centuries of breeding to select phenotypes that are controlled by combinations of genes, typically with undefined mutations, which produce the desired traits. Changes in the plant characteristics (e.g., disease resistance, insect resistance, food quality) typically have been slow, except when partial or full genomes have been combined. This combination as is hypothesized to have occurred thousands of years ago in the generation of modern hexaploid wheat as a hybrid cross of tetraploid and diploid progenitors (1). Such crosses result in the combination of hundreds to thousands of different proteins in a single food. In contrast, modern tools of biotechnology have allowed the introduction of one or a few new genes and resulting proteins into crop varieties that are carefully selected and studied for safety and performance before being allowed into commercial production (2, 3). As methods to introduce desired traits in plants have improved, so too has the public

Purification, characterization and safety assessment of the introduced cold shock protein B in DroughtGard maize.

DroughtGard maize was developed through constitutive expression of cold shock protein B (CSPB) from Bacillus subtilis to improve performance of maize (Zea mays) under water-limited conditions. B. subtilis commonly occurs in fermented foods and CSPB has a history of safe use. Safety studies were performed to further evaluate safety of CSPB introduced into maize. CSPB was compared to proteins found in current allergen and protein toxin databases and there are no sequence similarities between CSPB and known allergens or toxins. In order to validate the use of Escherichia coli-derived CSPB in other safety studies, physicochemical and functional characterization confirmed that the CSPB produced by DroughtGard possesses comparable molecular weight, immunoreactivity, and functional activity to CSPB produced from E. coli and that neither is glycosylated. CSPB was completely digested with sequential exposure to pepsin and pancreatin for 2 min and 30 s, respectively, suggesting that CSPB will be degraded in the mammalian digestive tract and would not be expected to be allergenic. Mice orally dosed with CSPB at 2160 mg/kg, followed by analysis of body weight gains, food consumption and clinical observations, showed no discernible adverse effects. This comprehensive safety assessment indicated that the CSPB protein from DroughtGard is safe for food and feed consumption.