Purification and molecular cloning of a major allergen from Anisakis simplex

A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.