Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells

• Published in: Biological chemistry Vol. 382; no. 12; pp. 1669 - 1677
• Main Authors: Camaj, P, Hirsh, A E, Schmidt, W, Meinke, A, von Gabain, A
• Format: Journal Article
• Language: English
• Published: Germany 01.12.2001
• Subjects: Amino Acid Sequence; Bacterial Outer Membrane Proteins - immunology; Bacterial Outer Membrane Proteins - metabolism; Bacteriophage T7 - immunology; Bacteriophage T7 - metabolism; Epitopes - metabolism; Escherichia coli - immunology; Escherichia coli - metabolism; Flow Cytometry; Gene Library; Immunomagnetic Separation; Ligands; Molecular Sequence Data
• ISSN: 1431-6730
• DOI: 10.1515/BC.2001.202

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 plafform 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.