American cockroach (Periplaneta americana) is a major source of indoor allergens in tropic and sub-tropical regions, frequently causes allergic reactions and asthma. Since no dominant major allergen exists in this source, component resolved diagnosis (CRD) is required to determine patients’ sensitization and pointing out treatment options. Therefore, the ERA-Net New Indigo project GENALL (Genetically engineered allergens for component-resolved diagnosis and immunotherapy of airway allergies) aimed to produce a set of well-characterized recombinant Periplaneta americana allergens to establish CRD.
Per a 1.0103 (residues 197 – 378) a microvillar membrane associated protein homologue, Per a 2.0101 an unusual aspartic protease, and the C-terminal domain of Per a 3.0101 (residues 426 - 675), a hemocyanin, were produced recombinantly in E.coli. The primary sequence of purified proteins was confirmed by amino acid analysis (AAA) and mass spectrometry (MS). Secondary structure elements were determined by circular dichroism spectroscopy (CD) and proteins were analyzed for aggregation behavior. Models of the proteins were generated with Swiss-Model using homologues Blattella germanica allergens as templates. Sera from cockroach allergic patients from India and South Korea were used to test IgE sensitization to these proteins in ELISA.
The identity of the three purified proteins was confirmed by MS and concentrations ranging from 0.3 - 1 mg/ml were determined by AAA. CD revealed partially intact secondary structural elements of the proteins, resembling the modeled structures. While for Per a 2 and Per a 3 a sensitization frequency of 50 – 92% was observed in ELISA experiments, reactivity to Per a 1 was limited in the tested patients’ cohort.
Purified, recombinant Per a 1, Per a 2 and Per a 3 were physico-chemically characterized and their ability to bind IgE was shown. By including all currently described Periplaneta americana allergens and testing diverse patients’ cohorts from Europe, Asia and America, the sensitization pattern of these allergens can be elucidated.