title: Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria.
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 degrees 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 degrees 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-Ralpha 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.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Ramat Aviv 69978, Israel. benhar@post.tau.ac.il
authors.forename: I
authors.initials: I
authors.lastname: Benhar
authors.forename: R
authors.initials: R
authors.lastname: Azriel
authors.forename: L
authors.initials: L
authors.lastname: Nahary
authors.forename: S
authors.initials: S
authors.lastname: Shaky
authors.forename: Y
authors.initials: Y
authors.lastname: Berdichevsky
authors.forename: A
authors.initials: A
authors.lastname: Tamarkin
authors.forename: W
authors.initials: W
authors.lastname: Wels
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000235:genetics / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000502:physiology / Q000821:virology; D029968:Escherichia coli Proteins; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000502:physiology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008074:Lipoproteins; D019151:Peptide Library; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D000906:Antibodies; D000941:Antigens; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D008074:Lipoproteins; D019151:Peptide Library; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; C099932:Lpp protein, E coli; C000715470:MAS1 protein, human; C078167:OMPA outer membrane proteins