Selection of beta-lactamase on filamentous bacteriophage by catalytic activity.
Recently the display of repertoires of peptides and proteins on the surface of filamentous phage, and selection of the phage by binding to a ligand, has allowed the isolation of peptides and proteins with rare binding activities. Furthermore, phages displaying enzymes (phage enzymes) have been selected by affinity of binding to inhibitors. Here we show, using a suicide inhibitor, that phage enzymes can also be selected by their catalytic activity. Two phage enzymes were constructed by fusion to the minor coat protein of the phage (g3p), displaying either an active beta-lactamase or a catalytically inactive mutant in which the essential serine of the active site was mutated to alanine. The phages were then incubated with a beta-lactamase suicide inhibitor connected by a spacer to a biotin moiety. The active (but not the inactive) phages were labelled, and the active phages selected from mixtures with inactive phages by binding and elution from streptavidin-coated beads. The selection ratio for active versus inactive phages (about ten on elution of the phages by reduction of an S-S bond in the spacer between the warhead and biotin) could be improved to about 50 on elution by proteolytic cleavage of beta-lactamase from g3p at an intervening factor X site. Selection of phage-enzymes by catalysis may provide a means of creating new enzymes and refining their catalytic properties.
Soumillion
P
P
Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Belgium.
Jespers
L
L
Bouchet
M
M
Marchand-Brynaert
J
J
Winter
G
G
Fastrez
J
J
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin; D002384:Catalysis; D017931:DNA Primers; D005170:Factor X / Q000096:biosynthesis / Q000378:metabolism; D005814:Genes, Viral; D007202:Indicators and Reagents; D017912:Inovirus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D012694:Serine; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D007202:Indicators and Reagents; D008024:Ligands; D012694:Serine; D001710:Biotin; D005170:Factor X; D001618:beta-Lactamases; D000409:Alanine