Biomedical Engineering Reference
In-Depth Information
domain is fused to the N-terminus of a second 'prey' protein (Figure 8.1). Once fragmented,
the two parts of the GAL4 protein can no longer drive transcription of GAL4-dependent
genes. However, if the BD and AD domains are fused to proteins that interact, then the
GAL4 BD and AD domains are brought into close enough proximity to drive transcription
of an adjacent downstream reporter gene. In most contemporary Y2H systems, a combi-
nation of biosynthetic and enzymatic reporter genes are used to minimize the possibility
of promoter-specific false-positive results [5]. In the Y2H system described in this chapter,
three reporter genes are used to detect positive interactions. These include two biosyn-
thetic reporter genes (ADE2 and HIS3) and one enzymatic (lacZ) reporter gene, each of
which is under the control of different Gal4-induced promoters (Gal2p, Gal1p and Gal7p
respectively).
Although the basic principles of all Y2H methods are similar, several different Y2H
systems have now been developed. Therefore, it is important to emphasize that vectors and
host strains from different Y2H systems may not be compatible. Care should be taken to
check both the genotype of the host strain(s) being used and the characteristics of different
bait and prey vectors. There can be considerable variations in the amount of bait or prey
fusion protein expressed in different GAL4-derived Y2H systems. These differences can
have dramatic effects on the strength and number of interactions detected under equivalent
selection conditions. For example, the pGAD prey vector has a less efficient promoter than
that found in pACT vectors. As such, fewer interactions will be detected with a pGAD
vector. However, the high stringency of the pGAD system means that interactions that are
detected may be strong. At times lower stringency may be required to detect very weak
or transient interactions. In this case pACT-derived vectors may be a more appropriate
choice if appropriate controls are used. The key point is to fully understand the inherent
characteristics of the system being used.
8.2 Methods and approaches
The procedures described in this chapter have been developed to provide sensitive medium
or high-throughput assays, which, in combination with stringent controls, can provide large
amounts of high-confidence binary protein interaction data. In essence, they utilize the
PJ69-4A yeast strain developed by James
et al
. [5], a set of bait and prey recombination
vectors developed by Semple and Markie [11] and a selection of high-throughput adap-
tations inspired by the pioneering work of Walhout and Vidal [12]. Adaptations used in
the procedures described in this chapter have the advantage that a single polymerase chain
reaction (PCR) product can be used to generate either bait or prey clones, there is no need
for the use of expensive cloning enzymes, and vectors are compatible with both Gateway
TM
format inserts and a large collection of commercially available Y2H libraries (Clontech).
When performing medium- or large-scale Y2H studies, three considerations must be
taken into account: the production of large numbers of bait and/or prey constructs; the
rapid screening of potential interactions; and the use of appropriate controls to eliminate
false-positive data. These criteria are equally valid for both targeted 'matrix-style' Y2H
studies or the screening of high-complexity Y2H libraries.
8.2.1 Producing large numbers of bait or prey clones
The process of producing large numbers of bait and prey clones can be expedited by the
use of
in vivo
Gap repair cloning methods [13, 14]. This approach is based on the simple
