Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays.
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.
Kodzius
Rimantas
R
Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, CH-7270 Davos, Switzerland.
Rhyner
Claudio
C
Konthur
Zoltan
Z
Buczek
Donald
D
Lehrach
Hans
H
Walter
Gerald
G
Crameri
Reto
R
D000485:Allergens / Q000737:chemistry*; D001232:Aspergillus fumigatus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D019151:Peptide Library; D014176:Protein Biosynthesis; D017455:Receptors, IgE; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000485:Allergens; D018076:DNA, Complementary; D019151:Peptide Library; D017455:Receptors, IgE