Purification and characterization of natural Ara h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform

The peanut allergen Ara h 8 is an important allergen for birch pollen allergic patients because of the cross-reactivity to the homologous Bet v 1. As the existence of Ara h 8 has been shown at the cDNA level so far (AY328088) and the allergen has indirectly been detected as natural protein, it was the aim of our study to identify natural Ara h 8 in peanut extract and to develop a purification strategy. This was achieved using a unique combination of purification steps, including optimized extraction conditions, size exclusion and ion exchange chromatography and treatment of the interfering contaminants with iodoacetic acid. A characterization of the protein by microsequencing showed discrepancies to the deduced amino acid sequence of AY328088. For this reason, we cloned and expressed a new Ara h 8 isoform from cDNA (EU046325). This IgE-reactive protein corresponds to the results of microsequencing, ESI-FTICR-MS and trypsin fingerprinting analysis of the authentic and purified nAra h 8. Apart from the ultimate use of recombinant allergens for diagnostic procedures, there is also a scientific need for the natural counterpart, as it represents an excellent reference point by which to compare protein characteristics and to standardize diagnostic and therapeutic allergens.