Description: Phage display describes a selection technique in which a library of variants of a peptide or protein is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside (1-3). This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called panning (4). In its simplest form (Figure 1), panning is carried out by incubating a library of phage-displayed peptides with a plate (or bead) coated with the target, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences. After 3-4 rounds, individual clones are characterized by DNA sequencing and ELISA.
New England Biolabs offers 3 pre-made random peptide libraries, as well as the cloning vector M13KE for construction of custom libraries. The pre-made libraries consist of linear heptapeptide (Ph.D.-7) and dodecapeptide (Ph.D.-12) libraries, as well as a disulfide-constrained heptapeptide (Ph.D.-C7C) library. The randomized segment of the Ph.D.-C7C library is flanked by a pair of cysteine residues, which are oxidized during phage assembly to a disulfide linkage, resulting in the displayed peptides being presented to the target as loops. All of the libraries have complexities in excess of 2 billion independent clones. The randomized peptide sequences in all three libraries are expressed at the N-terminus of the minor coat protein pIII, resulting in a valency of 5 copies of the displayed peptide per virion. In both the Ph.D.-7 and the Ph.D.-12 libraries, the first residue of the peptide-pIII fusion is the first randomized position, while the first randomized position in the Ph.D.-C7C library is preceded by Ala-Cys. All of the libraries contain a short linker sequence (Gly-Gly-Gly-Ser) between the displayed peptide and pIII.
The Ph.D. libraries have been used for myriad applications, including epitope mapping (Figure 2), identification of protein-protein contacts (5) and enzyme inhibitors (6) and discovery of peptide ligands for GroEL (7), HIV (8-11), semiconductor surfaces (12) and small-molecule fluorophores (13) and drugs (14). Bioactive receptor ligands have been identified both by panning against purified receptors (15-18) and against intact cells (19-22). Peptides which target specific cell types have been isolated by in vitro panning and used for cell-specific gene delivery (23-26). Ligands for mold spores (27) and bacterial cells (28) have also been identified using this system, including a peptide that specifically inhibits anthrax toxin, both in vitro and in vivo (29). Finally, tissue-specific peptides have been isolated by in vivo panning, in which phage is injected into a live animal, the relevant organs harvested and phage isolated from each tissue type (30,31).
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