Diversity of Phage-Displayed Libraries of Peptides during Panning and Amplification

Abstract

Abstract:The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins), it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells). This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional "phage phase diagram", which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of OPEN ACCESSMolecules 2011, 16 1777 amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

“…S4A). The identification of just hnRNPA2 by conventional phage display screening underscores one of its main limitations, namely the retrieval of very few candidates, likely because many bona fide targets fail to be properly amplified or selected through multiple biopanning rounds (33). The results here presented show that PD-Seq overcomes this shortcoming, providing an opportunity for the high-throughput identification of smallmolecule targets.…”

Deep sequencing analysis of phage libraries using Illumina platform

“…In previous publications, the result from sequencing of ~100 phage clones suggested that the amplification process enriches for specific peptide sequences [15,16]. Large scale sequencing, however, can provide observations that could not be interpreted from the sequence of 100 clones [17].…”

“…It has enabled the discovery of ligands for hundreds of targets, yet the literature still contains several poorly-explained observations: (1) identical sequences could emerge from unrelated screens for unrelated target [22,23], and (2) screens that should yield a large number of diverse ligand often yield only one sequence motif (reviewed in [16]). The nearly complete sequence coverage of libraries illuminates the origin of these observations.…”

Prediction of Protein Interaction Sites Using Mimotope Analysis

“…Phages with growth advantage can be not only noise but also decrease the library diversity and lead to a loss of useful mimotopes. Simulations and experiments showed that subtle differences in growth rate yielded drastic differences in clone abundances after rounds of amplifications (Derda, et al, 2011). Thus, propagation-related TUP may even dominate the biopanning results.…”

“…They appear in the biopanning results because they are selected by contaminants or other components of the screening system rather than the target (Menendez & Scott, 2005;Vodnik, et al, 2011). Propagation-related TUP makes another category (Brammer, et al, 2008;Derda, et al, 2011;Thomas, et al, 2010). They sneak into the output of biopanning because they have a higher infection rate or faster secretion rate.…”