Interaction analysis through proteomic phage display

Abstract

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

Figure 1

Schematic representation of proteomic phage display using the M13 phage. Input phage display libraries are constructed from cDNA, ORFs, or oligonucleotide arrays designed from a proteome of interest (1). Peptides are displayed on pVIII (2). Bait proteins are immobilized on a solid surface and incubated with the naïve input phage library (3). Binding of phage occurs through interactions between displayed peptides and bait proteins, but nonspecific interactions cause noise in the selection (not shown). Unbound phage is washed away (4) and bound phage is eluted through acidic or basic conditions or by the addition of actively growing host bacteria (5). Eluted phage is amplified (6) and used for repeated (typically 3–5) cycles of selection, which is necessary to amplify specifically bound phage over nonspecific binders. Sanger sequencing of confirmed binders and/or NGS of the retained phage pools provides lists of binders from the target proteome (7).

Figure 2

Schematic representation of the assembly and excretion of the M13 filamentous phages and the exit through cell lysis of the lytic T7 phage. (a) The filamentous M13 phage is assembled at the cell membrane of the bacterial host. In the host cell, the ssDNA is protected by association with protein pV, which detaches at the membrane upon assembly. At the start of the assembly, membrane associated pVII/pIX bind a specific DNA packing signal. Membrane bound protein pVIII binds to the DNA and is transferred across the membranes. The transport is facilitated by a complex of pI and pXI situated in the inner membrane and protein pIV that makes a pore through the outer membrane of the bacteria for the phage to pass through. As a final step, pVI and pIII that span the inner membrane are assembled on the phage. The figure was created based on [31]. (b) The lytic T7 phage, schematically shown with its typical icosahedral head, is assembled in the cytosol of the host cell. It is multiplied to such extent that the host cell finally bursts and the phage is released to the surrounding.