Biomedical Engineering Reference
In-Depth Information
trometry. Many variations of this approach exist, most notably among them one
that uses co-immunoprecipitation to isolate a complex of interest. Here, the tag
is a peptide epitope that can be recognized by an antibody, a feature that can be
used to render a protein complex insoluble.
To identify numerous protein complexes, it is necessary to repeat an ex-
periment of this kind with many different bait proteins. The largest-scale ap-
proaches so far have identified more than 500 protein complexes in the yeast
Saccharomyces cerevisiae
(13,16).
3.3. Protein Arrays (Protein "Chips")
A protein array consists of hundreds or thousands of well-defined locations
on a small surface, each of which contains many copies of one protein. Protein
arrays are useful not only to study protein-protein interactions, but also a variety
of other aspects of protein function. There are again several approaches (re-
viewed in (35)), of which I will present only one representative example, along
with one application (34).
In one approach to creating a protein array, a collection of many different
genes that encode proteins of interest is established. Each gene in the collection
is fused to the coding region of GST (see above) and to a stretch of DNA encod-
ing many histidine residues (PolyHis). Upon expression of these chimeric genes,
the fusion proteins can be attached to a glass slide via their PolyHis tail, each
protein in a separate and well-defined spot. (The glass slide constitutes the pro-
tein chip.)
Zhu and collaborators (34) used such protein arrays to ask which proteins in
the yeast
Saccharomyces cerevisiae
interact with calmodulin. Calmodulin is an
important protein in many organisms and mediates the action of calcium ions on
cells. It is, for example, involved in the secretion of proteins and in the motility
of vesicles inside cells. To identify which yeast proteins interact with
calmodulin thus means to identify which proteins contribute to regulating the
effects of Ca
2+
on yeast cells. The employed assay uses biotinylated calmodulin,
that is, calmodulin to which biotin has been attached. Biotin is a compound that
is very tightly and specifically bound by the protein streptavidin, its sole purpose
in this assay. Another important ingredient of the assay is streptavidin that is
chemically modified by attaching a green fluorescent dye, Cy3, to it. In the as-
say, the array is exposed to a solution of biotinylated calmodulin and Cy3-
labeled streptavidin. All proteins that interact with calmodulin will have bioti-
nylated calmodulin and Cy3-labeled streptavidin bound to them. The corre-
sponding spots on the array will appear green when exposed to light of the
appropriate wavelength.
