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

Comb Chem High Throughput Screen. 2003 Mar;6(2):147-54. doi: 10.2174/1386207033329751.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Allergens
  • DNA, Complementary
  • Peptide Library
  • Receptors, IgE