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 enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis 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 complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.
Demartis
S
S
Institut für Molekularbiologie und Biophysik, ETH Hönggerberg, Zürich, CH-8093, Switzerland.
Huber
A
A
Viti
F
F
Lozzi
L
L
Giovannoni
L
L
Neri
P
P
Winter
G
G
Neri
D
D
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry*; D001483:Base Sequence; D019921:Biotinylation; D002147:Calmodulin / Q000737:chemistry; D002148:Calmodulin-Binding Proteins / Q000737:chemistry; D002213:Capsid / Q000235:genetics; D002384:Catalysis; D011232:Chemical Precipitation; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000737:chemistry*; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008863:Microspheres; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism