Phage display of enzymes and in vitro selection for catalytic activity.
Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes. The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography. The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.
Soumillion
P
P
Laboratoire de Biochimie Physique et des Biopolyméres, Université Catholique de Louvain, Belgium.
Jespers
L
L
Bouchet
M
M
Marchand-Brynaert
J
J
Sartiaux
P
P
Fastrez
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000668:Ampicillin Resistance; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D017912:Inovirus / Q000235:genetics*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D013381:Subtilisins; D001618:beta-Lactamases