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

Rimantas Kodzius, Claudio Rhyner, Zoltan Konthur, Donald Buczek, Hans Lehrach, Gerald Walter, Reto Crameri*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)147-154
Number of pages8
JournalCombinatorial Chemistry and High Throughput Screening
Volume6
Issue number2
DOIs
StatePublished - Jan 1 2003

Keywords

  • Allergens
  • High-density arrays
  • Phage display
  • Robotics
  • cDNA

ASJC Scopus subject areas

  • Drug Discovery
  • Computer Science Applications
  • Organic Chemistry

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