Biomedical Engineering Reference
In-Depth Information
gene products is often regulated through post-translational modifications such as
glycosylation and phosphorylation. mRNA expression, by definition, cannot
resolve such differences. In contrast, protein expression studies can identify
some of them. For example, different glycosylation states of the same protein
manifest themselves in 2D electrophoresis as a closely spaced series of spots.
Similarly, protein phosphorylation has specific effects on the mass/charge signa-
ture of proteins that can be detected with electrophoretic and mass spectroscopic
approaches. However, post-translational modifications do pose enormous chal-
lenges to protein expression analysis, especially in light of the fact that hundreds
of chemical modifications are possible. In particular, often only a small fraction
of the same protein molecules are modified per cell, necessitating enormously
sensitive approaches to detect them. In addition, while a single peptide sequence
(obtained through tandem mass spectrometry) can be sufficient to identify a pro-
tein uniquely, this holds no longer for the identification of post-translational
modifications (1).
3.
IDENTIFYING PROTEIN INTERACTIONS
Protein networks in the strong sense are identified through approaches that
detect interactions of proteins with each other. I will briefly discuss three ap-
proaches to detect such protein interactions on a large scale, the yeast two-
hybrid assay, a second technique to identify protein complexes, and protein ar-
rays or protein "chips" (reviewed in (23)).
3.1. The Yeast Two-Hybrid Assay
As originally proposed (10), the yeast two-hybrid assay is a technique to
identify interactions between two specific proteins A and B (not necessarily
from yeast). It takes advantage of the fact that many eukaryotic transcriptional
activators consist of two functionally different and separable parts: a DNA bind-
ing domain responsible for binding of the transcription factor to DNA, and a
transcriptional activation domain responsible for interacting with RNA poly-
merase. Even when separated (e.g., attached to different polypeptides), these
domains can still exert their roles of DNA binding and transcriptional initiation.
To detect interactions between protein A and protein B, the two-hybrid as-
say first uses recombinant DNA techniques to generate two hybrid proteins. One
of them is a hybrid where protein A is fused to the transcriptional activation
domain of a yeast transcription factor, such as the gene product of
GAL4
. This
hybrid protein is also called the bait protein. The second hybrid protein is a pro-
tein where the transcriptional activation domain of the transcription factor is
fused to protein B. This hybrid protein is also called the prey (see also Figure 2).
