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Biol Chem. 2001 Dec;382(12):1669-77.

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

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  • 1InterCell Biomedizinische Forschungs- und Entwicklungs-AG, Vienna, Austria.

Abstract

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.

[PubMed - indexed for MEDLINE]
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