Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays

• Published in: Combinatorial chemistry & high throughput screening Vol. 6; no. 2; p. 147
• Main Authors: Kodzius, Rimantas, Rhyner, Claudio, Konthur, Zoltan, Buczek, Donald, Lehrach, Hans, Walter, Gerald, Crameri, Reto
• Format: Journal Article
• Language: English
• Published: United Arab Emirates 01-03-2003
• Subjects: Allergens - chemistry; Aspergillus fumigatus - metabolism; Cloning, Molecular; DNA, Complementary - chemistry; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Peptide Library; Protein Biosynthesis; Receptors, IgE; Reverse Transcriptase Polymerase Chain Reaction
• ISSN: 1386-2073
• DOI: 10.2174/1386207033329751

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 affinity-selected 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.