Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

Abstract

Abstract:We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus

“…More allergens (defined by IgE binding) have been characterized from A. fumigatus than from all other fungal species combined (n ¼ 58) 12 . We identified nine additional predicted allergens in the genome based on similarity with other fungal allergens (Supplementary Table S3), including secreted proteases, glucanases and cellulases.…”

Vaccinations with Recombinant Variants of Aspergillus fumigatus Allergen Asp f 3 Protect Mice against Invasive Aspergillosis

“…Similar truncated versions were reported to lack the ability to bind human IgE from ABPA patients (37). Accordingly, such engineered proteins no longer possess the IgE-binding property by which most A. fumigatus allergens have been defined (24). IgG2a from sera of HSimmunized animals reacts with full-length rAsp f 3 but not with the truncated versions, suggesting that the IgG2a epitope responded to in these mice might be similar (if not identical) to the IgE-binding conformational epitope in sera from ABPA patients (37).…”

The GPI‐anchored Gas and Crh families are fungal antigens

“…The gbAF062651 (Aspf16) corresponds to CRF1 (98% identity) but includes several sequencing errors, three of them leading to different frame-shifts (Banerjee et al, 2001). The sequence of rAsp f9 cDNA (AFAJ3227) corresponded to a fragment of the CRF1 cDNA, the sequence this fragment being 100% identical to that of rAsp f9 (Crameri et al, 20 001;Kodzius et al, 2003). While the full-length CRF1 cDNA encodes a protein of 395 amino acids, the rAsp f9 cDNA codes for a protein of 302 (lacking 93 amino acids from the carboxyterminal).…”