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 thei r properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants e ndowed with the anticipated properties from a large collection of poss ible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalyti c activity of the encoded enzymes. The gene coding for the enzyme to b e engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzym e. 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 phage s showing an activity on the inhibitor under the conditions of the exp eriment are labeled. These phages can be recovered by affinity chromat ography. The design of the label and the factors controlling the selec tivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discu ssed.