Biology Reference
In-Depth Information
gene. Since its beginnings, the yeast two-hybrid technology has been
widely used for screening interactions in a number of organisms
(including the large proteomes of higher eukaryotes). A number of
improvements and modifications have been made to the original pro-
cedure, including the use of shorter gene fragments and multi-purpose
versatile cloning vectors such as the Gateway system. In addition, vari-
ants of the method have been devised that work in cellular localizations
other than the nucleus, for example, in the cytoplasm or at the plasma
membrane.
11-14
While very powerful, yeast two-hybrid screens sometimes suffer from
lack of reproducibility between laboratories, and often exhibit limited
overlap with previously known protein-protein interactions. This is partly
explained by the “foreign” surroundings of the test proteins: these have
to fold correctly, and interact correctly, in an organism for which they
have not evolved, and often also in a cell compartment where they may
normally not be found (the nucleus). In addition, many protein-protein
interactions normally require more than two proteins — the formation of
protein complexes is thought to often involve cooperative binding and
may also require the action of chaperones and other assembly proteins,
most of which is presumably not possible inside the nucleus in a two-
hybrid screen.
Another widely used experimental technique for protein-protein
interaction detection is the biochemical purification of native protein
complexes, followed by the identification of their constituent proteins
using mass spectrometry (MS).
15,16
This is a very powerful technique, and
it has a number of advantages over two-hybrid techniques. First, entire
protein complexes that have been purified from their native surroundings
are analyzed. This enables the detection of complexes that may need an
elaborate series of steps for their assembly, and that may not form easily
outside their native surroundings. Second, biochemical purification of
the complexes can be performed based on specific and versatile peptide
epitopes, which are added to one protein of interest in a complex (the
“bait” protein). This means that several entry points (potential baits)
exist for each protein complex, so that the same complex can be purified
several times independently. The expression of bait proteins can also be
kept at endogenous levels, potentially keeping optimal stoichiometry by
