A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage

We have aimed at developing a general methodology for the isolation of enzy matic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage displa y, phage particles with suitable specificities are physically isolated by a ffinity capture and amplified by bacterial infection. Selection for catalys is mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complet e. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically iso lated using specific anti-product affinity reagents. We achieve the conditi onal anchoring of reaction substrates and products on phage by displaying e nzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions . Such phage particles can be targeted in a stable fashion (k(off) < 10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptid e-phage complexes are stable in purification procedures such as capture wit h magnetic beads and polyethylene glycol precipitation, and can be conditio nally dissociated by addition of calcium chelators. Glutathione-S-transfera se and an endopeptidase were used in model selection experiments to demonst rate that it is possible to isolate catalytic activities from calmodulin-ta gged enzymes displayed on filamentous phage, with enrichment factors >50 pe r round of selection. (C) 1999 Academic Press.