Since its development, the Y2H approach has now been applied
to an impressive variety of organisms and in various scale settings,
from pairwise to genomic level screens [4]. In particular, Y2H data
obtained with random cDNA libraries have opened the way to the
establishment of highly elaborate interactome maps within the
proteome of numerous species. However, in recent years the use
of smaller and arrayed libraries has been increasingly exploited.
These arrayed Y2H libraries can assemble proteins or domains
belonging to the same structural family or to diverse subcellular
entities. Their use can lead to a more comprehensive picture of the
interactions mediated by the array members [6–9]. Moreover, a
major advantage of arrays over random cDNA libraries when
screening by Y2H is their suitability for high-throughput technologies, making their implementation faster, more accessible, and
comprehensive.
Here, we describe the implementation of a Y2H screen that
focuses on PDZ-mediated interactions using an array-based human
PDZ ORFeome (thereafter named PDZome) generated in our
laboratory [10]. The PDZome holds 266 PDZ domains based on
the prediction and manual annotation of all the PDZ sequences.
New boundaries at the N- and C-termini have been established,
contributing to the folding, solubility, and affinity of the PDZ
domains [11]. As depicted in Fig. 2, candidate proteins known or
suspected to contain PDZ-binding motifs (PBMs) are used as baits
to screen the individual Y2H prey clones of the hPDZome array.
2 Materials
All solutions should be prepared using ultrapure water and autoclaved or sterile filtered. Store all reagents at room temperature
(unless indicated otherwise).
2.1 Design
of Primers for Bait
Constructs
The choice of a bait sequence to screen the PDZome library may be
guided by preliminary evidence of PDZ binding properties of a
candidate protein. When no information is available and because
most PDZ domains interact with the C-terminal end of their protein targets, a standard approach is to fuse the last fifteen residues of
a candidate protein in frame with the C-terminus of the Gal4-BD
moiety. In this case, generating the same bait but truncated of its
last three amino acids provides a convenient negative control as this
truncation is known to disrupt most PBM/PDZ interactions [12].
Of note, the use of larger cytosolic fragments may also prove
relevant when suspecting the occurrence of an internal noncanonical PBM within a given PDZ binding protein.
Below are guidelines for the design of primers for the cloning
of bait constructs using the Gateway
® cloning system. This cloning
strategy first requires the addition of attB1 and attB2
Y2H Screening of PDZ Interactions
3
to an impressive variety of organisms and in various scale settings,
from pairwise to genomic level screens [4]. In particular, Y2H data
obtained with random cDNA libraries have opened the way to the
establishment of highly elaborate interactome maps within the
proteome of numerous species. However, in recent years the use
of smaller and arrayed libraries has been increasingly exploited.
These arrayed Y2H libraries can assemble proteins or domains
belonging to the same structural family or to diverse subcellular
entities. Their use can lead to a more comprehensive picture of the
interactions mediated by the array members [6–9]. Moreover, a
major advantage of arrays over random cDNA libraries when
screening by Y2H is their suitability for high-throughput technologies, making their implementation faster, more accessible, and
comprehensive.
Here, we describe the implementation of a Y2H screen that
focuses on PDZ-mediated interactions using an array-based human
PDZ ORFeome (thereafter named PDZome) generated in our
laboratory [10]. The PDZome holds 266 PDZ domains based on
the prediction and manual annotation of all the PDZ sequences.
New boundaries at the N- and C-termini have been established,
contributing to the folding, solubility, and affinity of the PDZ
domains [11]. As depicted in Fig. 2, candidate proteins known or
suspected to contain PDZ-binding motifs (PBMs) are used as baits
to screen the individual Y2H prey clones of the hPDZome array.
2 Materials
All solutions should be prepared using ultrapure water and autoclaved or sterile filtered. Store all reagents at room temperature
(unless indicated otherwise).
2.1 Design
of Primers for Bait
Constructs
The choice of a bait sequence to screen the PDZome library may be
guided by preliminary evidence of PDZ binding properties of a
candidate protein. When no information is available and because
most PDZ domains interact with the C-terminal end of their protein targets, a standard approach is to fuse the last fifteen residues of
a candidate protein in frame with the C-terminus of the Gal4-BD
moiety. In this case, generating the same bait but truncated of its
last three amino acids provides a convenient negative control as this
truncation is known to disrupt most PBM/PDZ interactions [12].
Of note, the use of larger cytosolic fragments may also prove
relevant when suspecting the occurrence of an internal noncanonical PBM within a given PDZ binding protein.
Below are guidelines for the design of primers for the cloning
of bait constructs using the Gateway
® cloning system. This cloning
strategy first requires the addition of attB1 and attB2
Y2H Screening of PDZ Interactions
3