Target Discovery and Validation Reviews and Protocols

342 Cekaite, Hovig, and Sioud
nonrandom. High-throughput analysis of the motifs revealed similarities to
ligands for differentially expressed cell surface proteins, and a candidate ligand–receptor
pair was identified and validated. These data represent a step
toward the construction of a molecular map of human vasculature, and they
may have broad implications for the development of targeted therapies.
Phage display is a well established technology that allows detection and
characterization of protein–protein interactions. However, screening of large
number of clones is labor-intensive. Previously, we have demonstrated a proof
of principle of implementation of phage-displaying libraries with microarrays.
The recombinant phages were used for fabricating phage arrays that were further
screened against either breast cancer or healthy donor serum antibodies. A
significant tumor effect was found with most of the selected phage-displayed
peptides, suggesting that recombinant phage microarrays can serve as a tool in
monitoring humoral responses toward phage-displayed peptides (23). As shown
in Fig. 1, the immunoreactivity clusters of patients and healthy donors are
distinguishable, indicating that recombinant phage microarrays can monitor
humoral immune response in patients.
1.3. Ribosome Display
Ribosome display is an in vitro protein display system. The concept is to link
individual proteins to their genes (“phenotype linked to genotype”) and to create
libraries of such linkages. Similar to phage display, selection of a protein from
the library simultaneously captures its encoding gene, which can then be
expressed or manipulated. Key features of ribosome display are that it is a
wholly cell-free technology that can generate exceptionally large protein libraries
in vitro. The link between protein and gene (mRNA) is made through the ribosome
as a protein–ribosome–mRNA complex (48,49). DNA elements essential
for transcription and translation such as T7 promoter sequence, translation initiation
site, and immobilization tag sequence are added through PCR assembly
of DNA fragments. A coupled in vitro transcription and translation system
(such as rabbit reticulocyte lysate) is used to produce individual tagged proteins
on a tag-binding surface, which then are screened for binding to other proteins
such as enzymes and antibodies.
1.4. Conclusions and Future Challenges
Priming of the humoral arm of the immune system by tumor cells, other
cancer-causing agents, or both is printed in patient sera as a specific antibody
response. Therefore, probing of such humoral immune responses could provide
an effective strategy for cancer screening and therapy. Cancer antigens that may
be accessible to immune system at some stage of the disease may be used for
identification, classification, choice of therapy, or a combination.