Biomedical Engineering Reference
In-Depth Information
Using Yeast Two-Hybrid Methods
to Investigate Large Numbers
of Binary Protein Interactions
Panagoula Charalabous, Jonathan Woodsmith and Christopher M. Sanderson
Department of Physiology, School of Biomedical Sciences, University of Liverpool,
Liverpool, UK
8.1 Introduction
The yeast two-hybrid (Y2H) system is a well established procedure which detects direct
or 'binary' protein - protein interactions. Since its development in 1989 [1], the classic
Y2H assay has undergone a series of adaptations to increase stringency and reduce the
occurrence of false-positive interactions [2 - 5]. In recent years, new procedures have also
been developed to facilitate high-throughput analysis of many thousands of potential protein
interactions [6 - 10]. Although global 'interactome' projects often incorporate automated
robotic procedures, it is possible to perform thousands of Y2H assays, using relatively
inexpensive manual techniques. In the post-genomic era there is an increasing demand for
established procedures which can be used to provide a more extensive insight into the
organization and complexity of biological and pathological processes. In this chapter we
describe a series of reagents and procedures which can be used in any laboratory to perform
large-scale manual Y2H experiments.
The classical Y2H system [1] utilizes the inherent properties of the GAL4 transcription
factor, which is composed of two functionally distinct regions: a DNA binding domain
(BD) and a transcriptional activation domain (AD). The BD region binds to GAL4-specific
sequences in the promoter region of particular target genes. In contrast, the AD domain
drives transcription of adjacent downstream genes. In the classic Y2H assay the GAL4 pro-
tein is fragmented into its component domains. Conventionally, the BD region is fused to
the N-terminus of a protein or domain of interest to generate a 'bait' protein, while the AD
