Ligand-Mediated Protection against Phage Lysis as a Positive Selection Strategy for the Enrichment of Epitopes Displayed on the Surface of E. coli Cells

摘要

We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7·tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7·tag antibody was used as protective ligand for T7·tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10 000 cells displaying the T7·tag and approximately 10~8 cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7·tag displaying cells was observed, yielding an enrichment factor of up to 10~7-fold. The CISTEM technology was used to select sequences from a T7·tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display platform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.