Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria

Itai Benhar, Ronit Azriel, Limor Nahary, Shelly Shaky, Yevgeny Berdichevsky, Aviva Tamarkin, Winfried Wels

Research output: Contribution to journalArticlepeer-review

Abstract

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F+ Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F- by growth at 16°C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37°C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Rα chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries. Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)893-904
Number of pages12
JournalJournal of Molecular Biology
Volume301
Issue number4
DOIs
StatePublished - 25 Aug 2000

Keywords

  • Bacterial surface display
  • DIP selection
  • Lpp-OmpA' fusion
  • Phage display

Fingerprint

Dive into the research topics of 'Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria'. Together they form a unique fingerprint.

Cite this