Production of a recombinant protein from Alternaria containing the reported N-terminal of the Alt a1 allergen

Abstract

Allergen content of extract derived from Alternaria is somewhat variable. Allergenic molecules from Alternaria that appear as differing molecular size bands on IgE probed immunoblots may have a great deal of sequence homology and differ only in the length of the amino acid chain. One method to study this problem is to produce recombinant proteins from Alternaria. To explore these possibilities, the following experiments were performed. A strain of Alternaria was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration. Rapidly expanding mycelia were removed from the culture and mRNA was extracted. Purified mRNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification. Modified DNA was then spliced into lambda gt11 phage and yielded a recombinant library with 105 PFU. The library was screened for the presence of allergenic proteins using IgE containing human sera from Alternaria-sensitive patients. Positive plaques were cloned. PCR analysis of positive clones using an oligonucleotide from the reported N- terminal sequence of Alt a1 indicated an insert of 295 base pairs. Sequence analysis yielded a reading frame containing 84 amino acids and confirmed that this segment contained the code for the reported N-terminal amino acid sequence of Alt a1. A computer search for this sequence found no homologous proteins in the Entrez® sequences. Northern blotting studies on RNA purified from nine strains of Alternaria with the radiolabeled 247 BP DNA fragment indicated that this sequence was present in all strains. The 247 BP nucleotide was spliced into the Pflag® vector and clones containing insert in the proper reading frame were identified. The presence of recombinant protein in the clones was verified by SDSPAGE time studies. Protein produced in time studies was shown by immunoblotting and sandwich E1A to bind human IgE from Alternaria sensitive patients. This recombinant protein, containing amino acid sequence for Alt a1, is bound by human IgE and therefore should be useful as a model for studying allergy to the native Alternaria glycoprotein. Further research should define where this sequence occurs in the Alternaria genome and should determine the sequence of the entire protein.