title: Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions.
abstract: Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.
authors.affiliation: Biochemisches Institut, Universität Zürich, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Krebber
authors.forename: S
authors.initials: S
authors.lastname: Spada
authors.forename: D
authors.initials: D
authors.lastname: Desplancq
authors.forename: A
authors.initials: A
authors.lastname: Krebber
authors.forename: L
authors.initials: L
authors.lastname: Ge
authors.forename: A
authors.initials: A
authors.lastname: Pluckthun
mesh_terms: D002122:Calcium Chloride / Q000494:pharmacology; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000821:virology; D019793:Fluorescein; D005452:Fluoresceins; D005822:Genetic Vectors / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D017912:Inovirus / Q000235:genetics / Q000472:pathogenicity; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D019151:Peptide Library; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D005452:Fluoresceins; D007128:Immunoglobulin Fragments; D008024:Ligands; D019151:Peptide Library; D011506:Proteins; D014760:Viral Fusion Proteins; C055966:immunoglobulin Fv; D015636:Magnesium Chloride; D001618:beta-Lactamases; D002122:Calcium Chloride; D019793:Fluorescein