Synthetic Morphogens and Pro-morphogens for Aided Tissue Regeneration - Biologically Responsive Biomaterials for Tissue Engineering - page 52

3.7.1 Identi fi cation of Bioactive Sequences by Phage Displayed

Peptide Technique

Phage displayed peptide technique has been the main technological tool to identify
peptide sequences able to recognise cell receptors [ 33 ] . Phage displayed peptide
technology is based on the ability of bacteriophage to express foreign (poly)pep-
tides as fused sequences to the proteins of their capsid surface. This technique was
described for the first time by George P. Smith [ 34 ] . The display of speci fi c peptides
on the bacteriophage capsid surface is obtained by encoding a gene for a specific
peptide into the gene for a capsid structural protein. As a result the phage will syn-
thesise billions of pooled peptides which will be presented on phage particles to
constitute a phage-displayed peptide library. Unlike other methods, as many as
1,010 different peptides can be screened simultaneously for a targeted activity [ 35 ] .
The selection of peptides from these phage libraries focuses on the interaction with
proteins and enzymes often to interfere with their activity [ 36 ] . Since its discovery,
this technique has been used to design new agonists and antagonists for cell recep-
tors [ 37 ], for the development of vaccines and for the selection of new antibodies
[ 38, 39 ] as well as for improving the analysis of protein-to-protein interaction in
proteomics [ 40 ] and to identify new substrates and inhibitors in enzymology [ 41 ] .
Phage-displayed peptide libraries have been obtained by either lytic or filamentous
phages or by phagemid vectors [ 42 ] .
In the case of targeting of cell receptors, biopanning is considered the method
of choice as, from the initial bacteriophage pool, it provides small numbers of
clones, each one of them being specific for a single peptide and offering the desired
properties in terms of affinity or activity [ 33 ]. To select the peptide affinity the
phages are introduced to an immobilised target, the unbound phages are then
removed by washing and a final elution step allows the removal of the bound
phages. Typically, to ensure the selection of the high-affinity clones a few washing
and elution steps are performed. Once eluted, the specific phages are amplified in
host bacteria and undergo cyclical selection steps under more defined conditions
that signi fi cantly enhance their speci fi city [ 35 ]. In some cases, a negative selection
step can also be performed to increase the efficiency of the selection process. Once
the selection process has been completed, the primary structure of the displayed
peptide is easily identified by determining the nucleotide sequence of the corre-
sponding insert [ 35 ] .

3.7.1.1 Identi fi cation of Growth Factor Analogues

Recently, phage displayed peptide technique has allowed the identification of the struc-
ture and function of a number of cytokines and growth factors and their receptors [ 43 ] .
The restricted sequences of the OP-1 and several sequences of the VEGF have
been identified alongside with VEGF blockers and VEGF receptors 1 and 2 antago-
nists. In addition, sequences mimicking the PDGF-BB, NGF and IGF-1 have been