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

Delayed infectivity panning (DIP) is a novel approach for the in vivo isola tion of interacting protein pairs. DIP combines phage display and cell surf ace display of polypeptides as follows: an antigen is displayed in many cop ies 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 a re rendered functionally F- by growth at 16 degrees C. The antigen-displayi ng cells are used to capture antibody-displaying phage by virtue of the ant ibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 deg rees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scF v antibodies and the human ErbB2 proto-oncogene and IL2-R alpha chain as mo del antibody-antigen pairs, we demonstrate enrichment of those phage that d isplay a specific antibody over phage that display an irrelevant antibody o f over 1,000,000 in a single DIP cycle. We further show the successful isol ation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthe tic human library. The effectiveness of DF makes it suitable for the isolat ion of rare clones present in large libraries. Since DLP 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.