Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus

Summary Background Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen‐specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp‐specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody‐binding capacity for specific immunotherapy. Aim The aim was to clone, express and characterize a full‐length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE‐mediated mediator release. Methods Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5′ RACE) was performed to obtain full‐length cDNA coding for Pen a 1. Using a gene‐specific primer, PCR was performed and full‐length cDNA was cloned and sequenced. Recombinant His‐tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody‐binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE‐mediated type I allergy. Results The deduced amino‐acid sequence was 284 residues long and amino‐acid sequence identities with allergenic and non‐allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had α‐helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild‐type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation. Conclusions Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component‐resolved diagnosis and the generation of modified shrimp tropomyosin for allergen‐specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp ex