Target Discovery and Validation Reviews and Protocols

336 Cekaite, Hovig, and Sioud
Yeast two-hybrid assays and phage display are classical approaches for
systematic protein interaction screening capable of probing numerous interactions.
The principle of the yeast two-hybrid technique (1) is based on chimeric
proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a
protein “X” and a GAL4-activating region fused to a protein “Y”. If X and Y
can form protein–protein complex and reconstitute proximity of the GAL4
domains, transcription of a regulated marker gene occurs. The first genomewide
two-hybrid screen was performed by Bartel and co-workers for the study
of protein interactions in bacteriophage T7 (2). The first genome-wide studies
of a eukaryotic organism were performed by Uetz and co-workers and Ito and
colleagues in Saccharomyces cerevisiae (3,4). Also, a three-hybrid approach for
scanning the proteome for targets of small-molecule kinase inhibitors have been
developed (5).
Another strategy was introduced by G. P. Smith (6). It uses filamentous M13
bacteriophages. A single binding molecule may be displayed as a protein or
peptide fused to the surface of a bacteriophage and identified by sequencing the
encoding genome. Originally, phage display technology has been used for
selection of ligands for well characterized pure target proteins (e.g., monoclonal
antibodies and cell surface receptors) immobilized on a solid surface (7–9). In
this respect, a large number of peptides and single-chain Fv antibodies have
been selected for their ability bind various target proteins (10,11). Furthermore,
phage display techniques have been applied in profiling immune responses in
patients with autoimmune disease or cancers, and peptides or proteins binding
serum antibodies were identified (9,12–20). Moreover, cancer cell binding peptides
were selected from random peptide phage libraries (21).
An advantage of two-hybrid assays and phage display techniques is that no
previous knowledge about the interacting proteins/receptors is necessary for a
screen to be performed. Moreover, two-hybrid screening can be done in a
colony array format, in which each colony expresses a defined pair of proteins
(22). Recently, we have demonstrated that a high-throughput analysis of the
immune response in cancer patients can be performed by phage-based microarray
technique (18).
1.2. Examples of Protein Microarrays
Arrays of proteins have been used for both analysis of protein–protein
interactions and biochemical analysis of protein functions. The low volume
requirement and the multiplexed detection capability of microarrays enable
optimal use of precious clinical samples. In addition, the assays are rapid and
amenable to automation, thus large sample sets, as required for biomarker
studies, can be processed and analyzed. Also, through the use of optimized
detection methods and rigorous quality control, the assays can be very sensitive,