Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells.
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.
Camaj
P
P
InterCell Biomedizinische Forschungs- und Entwicklungs-AG, Vienna, Austria.
Hirsh
A E
AE
Schmidt
W
W
Meinke
A
A
von Gabain
A
A
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D017123:Bacteriophage T7 / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D015723:Gene Library; D018189:Immunomagnetic Separation; D008024:Ligands; D008969:Molecular Sequence Data
D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D008024:Ligands; C078167:OMPA outer membrane proteins