Biology Reference
In-Depth Information
Chapter 18
Using the Yeast Two-Hybrid System to Identify
Protein-Protein Interactions
Edgar Rodríguez-Negrete , Eduardo R. Bejarano , and Araceli G. Castillo
Abstract
The yeast two-hybrid system is currently one of the most standardized protein interaction mapping techniques.
The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain
from the transcriptional activation domain of several transcription factors. The protein of interest (bait) is
fused to a DNA-binding domain, and complementary DNA (cDNA) library-encoded proteins are fused to
a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both
activities of the transcription factor are rejoined resulting in transcription from a reporter gene. Here, we
describe protocols to test interactions between two individual proteins and to look for novel interacting
partners by screening a single protein or domain against a library of other proteins using a GAL4 based
yeast two-hybrid system.
Key words
Two-hybrid system, Protein-protein interaction, Yeast, GAL4, Interactors, Interactome
1
Introduction
The yeast two-hybrid system (Y2H) is an in vivo method to deter-
mine whether two proteins interact [
1
-
3
]
.
It relies on the modular
nature of many site-specifi c transcriptional activators, which consist
of a DNA-binding domain (BD) and a transcriptional activation
domain (AD). The system is based on the observation that the two
domains need not be covalently linked and can be brought together
by the interaction of any two proteins. To determine whether two
proteins interact, both must be expressed fused to either BD or AD
in a yeast strain that contains one or more reporter genes with
binding sites for the DNA-binding domain. If the proteins, bait
and prey, interact they create a functional activator by bringing the
activation domain into close proximity with the DNA-binding
domain detectable by expression of reporter genes (Fig.
1
). The
technology can also be used to identify novel protein interactions
using a bait protein fused to BD and a plasmid library that expresses
cDNA-encoded proteins fused to a transcription activation domain.
