Phage Display, A Laboratory Manual, Cold Spring Harbor
- C. F. Barbas, D. R. Burton, J. K. Scott, G. J. Silverman
Phage display provides a high-throughput method for the selection of large libraries of peptides or proteins with desired binding or catalytic properties. Phage display can also be used to screen cell proteomes to identify proteins that carry out specific biological functions. Typically in phage selection, a library of phage competes to bind to a target molecule linked to a solid support, and clones with desired binding properties can be enriched with a few rounds of selection. Selection for desired catalytic activities by phage display is more challenging because affinity selection of the phage library needs to be coupled with substrate turnover by the phage-displayed enzymes. In this study, we developed a method for phage selection of enzymes based on their catalytic activities. We attached substrate molecules to phage so that phage-displayed enzymes could catalyze product formation on the phage surface. We then selected for product-attached phage by affinity binding so as to enrich phage particles that displayed catalytically active enzymes. We demonstrated the feasibility of this method by attaching the substrate peptide of biotin ligase BirA to phage with the protein-modification enzyme Sfp phosphopantetheinyl transferase. Phage-displayed BirA catalyzed biotin conjugation to the substrate peptide. After the reaction, we used immobilized streptavidin to select for phage attached with biotinylated peptides for efficient enrichment of BirA clones. Overall, we have shown that substrate-attached phage prepared by Sfp can be a useful platform to select for enzymatic activities in a phage library. The purpose of attaching substrate molecules to phage particles in catalysis-based phage selection is that once the enzyme catalyzes substrate turnover on the phage surface, the product molecules do not diffuse away but are still attached to the phage. The catalytically active phage can then be selected by affinity binding with the product molecules (Scheme 1A). One direct method of attaching substrate molecules to phage is to have the substrates linked to the Cys side chains exposed on the phage surface (Scheme 1B). However, it is preferable to have precise control over the number and location of the substrate molecules conjugated to the phage particle. This can be achieved by expressing peptides or protein domains with specific binding properties as fusions to phage capsid proteins. For example, an “acidic peptide” can be displayed on the phage surface as a fusion to the pIII protein of the M13 phage (Scheme 1C). The acidic peptide can then bind to a “basic peptide” linked to DNA or peptide molecules to anchor the substrates of DNA polymerase or protein kinases on the phage. Similarly, calmodulin fused to the pIII protein was used for substrate attachment to the phage based on the tight binding of calmodulin with calmodulin-binding peptides linked to substrate molecules (Scheme 1D). In another study, the substrate peptide of subtiligase was directly fused to the enzyme displayed on the phage surface in order to select for improved ligase activities (Scheme 1E). Specific reactivity can also be programmed into the phage capsid proteins by incorporating SelenoCys into the pIII protein. SelenoCys offers a reactive seleno nucleophile to couple to substrate molecules with an a-iodo acetamide group (Scheme 1F). We recently developed a method for the codisplay of enzymes and peptide substrates on the phage surface and used this method to evolve the protein post-translational modification (PTM) enzyme Sfp. Sfp is a phosphopantetheinyl transferase that can modify an 11-residue peptide, ybbR, with smallmolecule probes conjugated to coenzyme A (CoA). To select for Sfp mutants that utilize 3’-dephospho CoA (dpCoA) in ybbR modification, we linked the coding sequence for the ybbR peptide to the pIII gene in the genome of M13KO7 helper phage to construct the helper phage “ybbR-M13”. ybbR-M13 produces phage particles with the ybbR peptide fused to the N terminus of the pIII capsid protein that is exposed on the phage surface (Scheme 1G). During phage proScheme 1. Various methods of attaching substrates to the phage surface. A) Scheme for the selection of catalytic activities with substrate-attached phage. B) Nonspecific attachment of substrate molecules to the phage surface. C) Acidic-basic peptide-mediated substrate attachment. D) Substrate attachment based on the binding between calmodulin and a calmodulin binding peptide. E) Substrate attachment by directly fusing the substrate peptide to phage-displayed enzymes. F) SelenoCys-mediated substrate attachment. G) Substrate attachment by fusing the substrate peptide to phage capsid protein pIII.