DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 23

can be useful. These serve to covalently cross-linking proteins that are
sufficiently close in space to be bridged by the cross-linker. This ap-
proach can work well to identify proteins can then be isolated from de-
tergent lysates of the cells and analyzed. The cross-linking agents are
frequently produced with a cleavable internal bond, such as a disulfide
bond, so that the two interacting proteins can be separated from one
another by reducing agents.
This approach can also be applied to proteins in cell lysates. In princi-
pal, the cross-linker is small enough to only span interacting molecules.
However, in a concentrated cell lysate, many proteins might be “nearby”
without being involved in functional interactions. To get around this
problem, purified bait proteins that are pre-reacted with one end of a
heterobifunctional cross-linked agents is often added to potential prey
proteins in cell lysates and allowed to interact. For these studies, the
other part of the bifunctional agent is not reactive until activated, usu-
ally by UV light. The interacting proteins are then analyzed to identify
potential prey.
Yeast two-hybrid screen
This is a very powerful genetic approach that not only allows an
investigator to determine if there are interacting partners for a known
bait protein, but also provides a genetic clone of the prey for easy
identification. Yeast two-hybrid screens are used for identifying a
variety of interacting proteins, including those that are transcriptional
activators. The beauty of the yeast two-hybrid system is that it can
be used to identify not only stably interacting proteins, but also those
proteins that associate only transiently in the cell.
The yeast two-hybrid system was first developed in 1989 (8) and
uses a bait protein to screen for an interacting protein(s) from a cDNA
library from the same species. Yeast (Saccharomyces cerevisiae) serves
as a host for the cDNA library screen. The yeast contains 2 plasmids,
one with the gene for the bait protein, and one that contains the cDNA
library (representing all the potential prey proteins). The system is set
up so that any protein that interacts with the bait will cause transcription
of a reporter gene, thus giving a visual readout of transcription, and
identifying a yeast clone that contains a cDNA that encodes a protein
that interacts with the bait.
The bait and prey cDNAs are expressed as fusion proteins with two
different protein domains that, when they interact, reconstitute a tran-
scriptional activation protein that drives a reporter gene. The Gal4 tran-
scription activation protein is the system of choice. Gal4 activates genes
encoding enzymes that are required by the yeast for galactose utilization.