Target Discovery and Validation Reviews and Protocols

Protein Arrays 343
The ideal proteomics-based analytical tool would consist of a microarray
containing a large number of high-affinity, high-specificity protein ligands. For
humans, this outcome would mean isolating on the order of 100,000 good
monoclonal antibodies or the equivalent capture proteins. In reality, the number
would be much higher, because the detection of different posttranslationally
modified forms of a protein is one of the principal advantages of moving
from nucleic acids to protein-based analytical techniques. However, the high
cost of monoclonal antibody manufacturing is a bottleneck for large-scale production
of antibody microarrays. Thus, it is most likely the field will proceed
in a stepwise manner, and a reasonable intermediate goal would be to construct
arrays of small amount of protein-binding ligands directed against proteins of
interest in a particular disease state. Reverse phase arrays and the aforementioned
display techniques allow the rapid and efficient isolation of high-affinity
and -specificity protein ligands. Furthermore, the uses of display techniques
allow identification of unknown biomarkers. It is increasingly evident that
posttranslational modifications of biomolecules could play an essential role in
coding for both new and native properties. However, the selection of antigens
with modifications is not accessible by peptide-on-plasmid or phage display
techniques. Regardless, the establishment of a robust system that may combine
the advantages of display strategies continues to be a key challenge. To achieve
certain desired display advantages, several different viral systems have been
used to display peptides, including lysogenic filamentous phages (6) and lytic
lambda phage (50,51), T7 bacteriophage (52), and T4 bacteriophage (53).
Lysogenic filamentous phages remain the most commonly used phage display
system. For cases in which displayed proteins may be toxic to filamentous
phage assembly or incompatible with the bacterial secretion pathway, lytic
phages can be used that allow displayed sequences to minimize negative
selection. However, selection of recombinant proteins displayed on T7 phage
by arrays seems to be difficult because negative phage immobilized on the
surface showed reactivity to patient antibodies as well (Cekaite et al., unpublished
data). One way of addressing this problem is to reduce binding valency by
including a binding competitor, thereby increasing the binding affinity threshold.
The examples in this chapter show that protein microarray technology is still
in its infancy, given the diversity of proposed approaches as well as raised problems.
However, this technology is promising tool for priming of the humoral
arm of the immune system.
References
1. Fields, S. and Song, O. -K. (1989) A novel genetic system to detect protein –protein
interactions. 340, 245–246.
2. Bartel, P. L., Roecklein, J. A., SenGupta, D., and Fields, S. (1996) A protein linkage
map of Escherichia coli bacteriophage T7. Nat Genet. 12, 72–77.