Introducation
The phage display technology represents a powerful tool for protein and drug design
because vast numbers of amino acid sequences can be rapidly explored. This review
describes the origins of phage libraries, their evolution and more recent advances,
including examples in the area of thrombosis and haemostasis, where the phage display
approach has just begun to be used with great success.
Phage display uses filamentous phages (E. coli specific phages with a filamentous shape that contain a single stranded closed circular
molecule of DNA), such as M13 or fd, as vehicles for displaying foreign peptides or
proteins on their surface. This is carried out by fusing the coding sequence (DNA)
for the peptide or protein to the amino (N)-terminus of either full-length phage minor
coat protein III (cpIII), or to phage major coat protein VIII (cpVIII), to carboxy
(C)-terminal domain of cpIII or, more recently, by fusion to the C-terminus of full-length
phage minor coat protein VI (cpVI) (Fig. 1). Expression of the fusion protein and
its subsequent incorporation into the mature phage particle results in the foreign
peptide or protein being presented on the phage surface. Thus, the linkage of each
peptide or protein to its encoding genetic material [contained as part of the single-stranded
viral DNA (1, 2)] represents a great advantage over conventional cloning methods.
The phage display approach was first used by Smith in 1985 (3), who expressed a library
of peptide sequences at the N-terminus of cpIII (3-5). This insertion allowed phage
assembly and display of the peptide on the phage surface, without affecting the phage
infectivity significantly. The linkage of genotype and phenotype in the phage library
allowed facile isolation of clones of specific interest from pools of millions of
clones by successive rounds of phage affinity selection on surfaces coated with a
ligand (panning) followed by phage amplification by infecting male E. coli (Fig. 2).
The foreign peptides or proteins were displayed, initially, on every copy of the coat
protein, but only short peptides can be displayed in this way without altering the
phage infectivity. Two systems have been developed to solve this problem. One incorporates
a second native gene III or VIII in the phage genome giving a mixture of native and
recombinant coat protein incorporation on the phage. The second system provides the
recombinant cpIII, cpVIII or cpVI gene on a phagemid (a plasmid containing the origin
of replication of filamentous phage). Phagemids can be packaged into phage particles
by superinfection with a helper phage. The fusion protein is incorporated onto the
surface coat, along with copies of the native coat protein encoded by the helper phage.
The result is a mixture of wild-type helper phage and recombinant phagemid particles,
but due to a defective origin of replication the helper phage is poorly packaged to
provide minimal competition with the phagemids (6). Phages that display both native
coat protein and fusion protein are infective. Therefore, the display systems can
be either multivalent or monovalent. Multivalent systems make use of gene III phage
constructs or gene VIII phage or phagemid constructs and give a high number of foreign
domains displayed on their surface (7). Monovalent systems utilize gene III or gene
VI phagemid constructs, which have a low number of foreign domains displayed on their
surface, usually a single copy. Consequently, monovalent systems distinguish between
low affinity and high affinity clones in panning assays (1, 8).
