Phage based screening strategy for identifying enzyme substrates

Abstract

Formylglycine generating enzyme (FGE) is conserved through most organisms for the post-translational formation of aldehyde bearing formylglycine residues, often at sites possessing the general motif XCXPXRX. Using a library of the XCXPXRX repertoire displayed on the p3 protein coat of fd phage, we devised a phage screening strategy for identifying FGE substrates. Our methods of screening phage possessing possible substrates for enzymatic activity made use of the unique aldehyde residues produced on the substrate by reaction with FGE, which facilitated covalent capture of phage onto magnetic beads. This sequence dependent conversion/capture in conjunction with an unexpected sequence dependence of the-enzymatic elution step used to liberate the phage for propagation served as the evolutionary selection pressures. The resulting HCTPRRP sequence identified from this phage screening procedure was an effective substrate for FGE, and through the course of the screening process it was selected for its propensity to be cleaved by trypsin. We anticipate this unique methodology for enzyme substrate screening may be applicable to selection of novel enzyme-substrate pairs by virtue of either sequence dependent immobilization or sequence dependent elution. (C) 2015 Elsevier B.V. All rights reserved.