View - ResearchGate

112 Casey et al.
be constructed by fusing DNA containing a degenerate region (typically using
NNG/T codons to minimize high frequency of stop codons) to a gene encoding
a coat protein usually gene III or gene VIII. This allows the foreign peptide
to be expressed as an N- or C-terminal fusion on the surface of the M13
bacteriophage (phage) coat protein. A large number of random peptide libraries
displayed on bacteriophage are now available, some are disulfide constrained by
inserting two cysteine residues, a typical library size ranges from 6 to 43 amino
acid residues (2). We chose to construct a 20-residue library, in order to select
for peptides long enough to permit short turns and other three-dimensional
structural features yet short enough to permit the production of a large diverse
library (3).
Selection of peptides of interest from the library that bind to a target molecule
can be performed using a process referred to as “panning.” This process allows
enrichment in binding affinity to the target molecule, and the resulting phage
peptides can easily be sequenced and further characterized. Peptides can be
synthesized without the phage framework and can be validated as separate
entities.
The advantages of peptide ligands for use in affinity chromatography include
the relative low cost of high quality stable peptides. In addition, instead of
having to prepare an affinity column using the whole recombinant antigen,
peptides that represent, for example, the antibody-binding site can be used. This
may also be beneficial as it may allow focus on relevant single specificities
and avoid unimportant epitopes if the whole protein was used for affinity
purification.
There are several applications of phage peptides that can be used for affinity
purification.
1. Peptide mimotopes: Phage mimicking the important epitopes of a given antigen
can be selected from a random phage peptide library by panning on antibodies
that bind to these epitopes. The peptides in principle could be useful for affinity
purification of antibodies specific for these important epitopes. These peptides
may be useful for serological monitoring of infectious diseases. Note: Definition of
peptide mimotopes: Peptides that bind to antibody-binding sites thereby mimicking
the three-dimensional conformational features of linear or conformational epitopes.
These peptides are defined as mimotopes as they mimic the essential features of
the epitope but do not necessarily bear sequence homology with the primary amino
acid sequence of the epitope (4).
2. Antigen-binding peptides: Another application of phage display for use in affinity
chromatography is the selection of a peptide that binds directly to an antigen.
These peptides could be useful for purification of the antigen itself. For example,
peptides of 5–7 residues flanked by two cysteines to form a disulfide bond were
selected from a phage display library, were immobilized onto a chromatographic