Phage display of enzymes and in vitro selection for catalytic activity

Abstract

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.

Engineering nitrile hydratase activity into a cysteine protease by a single mutation

“…Given the present unpredictability of the functional consequences of amino acid replacements, it is unlikely that protein engineering by itself can substitute for other methods to generate enzymes with novel activities. For the alteration of an enzyme that is not as well characterized, the use of random mutagenesis of the gene coding for the enzyme and phage display for the screening and selection of the desired activity offers an interesting alternative to the rational design approach (Clackson & Wells, 1994;Soumillion et al, 1994).…”

Selecting Open Reading Frames From DNA

“…Such an approach could be particularly useful in phage display, a technology originally developed to select peptide epitopes recognized by antibodies (Parmley andSmith 1988, 1989;Cwirla et al 1990;Balass et al 1993;Smith and Scott 1993;Yayon et al 1993), but subsequently expanded to include the display of antibodies (Marks et al 1991;Griffiths and Duncan 1998;Hoogenboom et al 1998) and many other proteins (for reviews, see Co et al 1991;Rada et al 1991;Saggio and Laufer 1993;Clackson and Wells 1994;Soumillion et al 1994;Bradbury and Cattaneo 1995;Choo and Klug 1995;Perham et al 1995;Burritt et al 1996;Cortese et al 1996;Iba and Kurosawa 1997;Lowman 1997). Although traditional phage display has been successfully applied to gene rich bacterial genomes (Jacobsson and Frykberg 1995Jacobsson et al 1997) and individual genes (Parmley and Smith 1989;Du Plessis et al 1995;Petersen et al 1995;Wang et al 1995;Bluthner et al 1996Bluthner et al , 1999, to identify antibody epitopes or binding partners, it suffers from the problem that only one clone in 18, if starting with DNA encoding an ORF, will be correctly in frame (one clone in three will start correctly, one clone in three will end correctly, and one clone in two will have the correct orientation), although experiments with synthetic peptide libraries have indicated that stop codons do not necessarily prevent display (Carcamo et al 1998).…”

Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants

“…Enzymes displayed on phage, for example, may be screened for improved affinity for desired substrates and the corresponding clones selected by panning techniques (22,25). Recently, a library of surface protease OmpT was displayed, and clones showing improved substrate affinity were isolated by flow cytometry (18).…”