Phage Display: Selecting Straws Instead of a Needle from a Haystack

Abstract

An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.

Identification of Immunotopes against <i>Mycobacterium leprae</i> as Immune Targets Using PhDTm- 12mer Phage Display Peptide Library

“…Display systems based on bacteriophages are widely used to assess different proteins and antibodies developed for identification of pathogenic bacteria [8][9][10]. A phage library that expresses random short peptide sequences on the surface of filamentous phage represents one of the biotechnological tools that could be helpful in amino acid sequence identification of various antigenic epitopes [11][12][13].…”

Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

“…The amino acid sequence of the peptide was identified using the ExPASy translate tool (http://web.expasy.org/translate/; Swiss Institute of Bioinformatics). All peptide sequences were screened using the online SAROTUP tool (15) to identify and eliminate any previously recognized target-unrelated peptide sequences (29).…”

Affinity Selection of Peptide Binders with Magnetic Beads <i>via</i> Organic Phase Separation (MOPS)

“…A well-known concern is that the amplification step during the biopanning leads to the loss of sequence diversity and real binding clones as well as to the increase of propagationrelated target-unrelated phage. 1,10) Reducing the number of amplification steps in biopanning is the only way to address these issues. 1) From this viewpoint, MOPS may be a good way for selecting peptide-binders.…”

“…1,10) Reducing the number of amplification steps in biopanning is the only way to address these issues. 1) From this viewpoint, MOPS may be a good way for selecting peptide-binders.…”

“…Conventional biopanning, however, sometimes leads to the isolation of false positive clones mainly due to their unspecific binding to components such as the solid phase, target-capturing molecules, blocking agents, etc. 1) Subtracting selection, where the background yield is reduced by preincubating the input phage with the solid phase coated with a capturing and blocking agent prior to incubation with target-coated solid phase, 2) and specific elution with known target ligands seem to be effective in removing target-unrelated phage.…”