Target Discovery and Validation Reviews and Protocols

Overview of Immune System 303
Fig. 11. Structure of the phage M13, showing minor and major coat proteins.
Recombinant peptides and proteins can be expressed as fusion with either pIII, pVI, or
pVIII coat proteins.
Peptides expressed on phages have a broad range of applications, which include
drug and target discovery, protein evolution, and rational design. DNA can
be cloned in frame at the amino terminus of the gene encoding for one of the
two-capsid proteins pIII or pVIII, and the corresponding fusion product will be
displayed on the surface of the virion (refs. 63–65; Fig. 11). Repertoires larger
than 10 8 phage clones expressing different peptide sequences can be prepared
from the host without lysis, giving very high titers of ligand-displaying phage.
Notably, phage display offers the advantage of the displayed ligand being
physically associated with the genetic information. Since the first reports, many
different peptide phage libraries have been constructed and screened with several
different ligates (65).
Because the integrity of the C terminus of pIII and pVIII is essential for efficient
phage assembly, insertions of foreign peptides can only be tolerated at the
N terminus. The use of phage for creating cDNA expression libraries has been
hampered by the presence of stop codons in the 3′ untranslated regions of
eukaryotic cDNA, which would prevent the fusion to the N terminus of the viral
coat proteins. To overcome this problem, Crameri and Suter (66) explored the
interaction between the Jun and Fos leucine zippers. Here, the Jun domain has
been expressed as a fusion with pIII and Fos expressed as an N-terminal fusion
peptide to a foreign protein. The resulting Fos-fusion protein associates with the
Jun-expressing phage particles. Jesper et al. (67) have described a novel phagemid
vector that allows the functional expression of full cDNA libraries as a fusion
with the C terminus of pVI protein. The phage coat protein pVI is not known to
be involved in infection, and its C terminus is thought to be surface exposed.
Therefore, the presence of stop codons in full-length cDNA does not prevent
display of cDNA repertoires fused to pVI. More importantly, the developed
cloning system allows the display in all three reading frames. We have explored
this expression system to identify tumor antigens (68).
A necessary step in filamentous phage assembly is the transport of fusion peptides
or proteins to the periplasm. This transport is a major limitation to phage