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.