A Combined Yeast/Bacteria Two-hybrid System: Development and Evaluation

Ilya G Serebriiskii,Rui Fang,Ekaterina Latypova,Richard Hopkins,Charles Vinson,J Keith Joung,Erica A Golemis

doi:10.1074/mcp.t500005-mcp200

Abstract

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.

Highlights

Yeast two-hybrid systems [1,2,3,4] are standard tools used to identify novel protein-protein interactions and to perform structure-function analysis on previously defined protein-protein interactions
In order to increase the power of a two-hybrid approach to identify and analyze protein interactions in high throughput applications, one approach has been to translate the basic components of the yeast two-hybrid system to a bacterial host organism [9,10]
Certain proteins can be problematic as baits in the yeast two-hybrid system; for example, proteins that are normally excluded from the nucleus in eukaryotes, that are potentially sequestered via interaction with an abundant partner evolutionarily conserved in yeast, or that stimulate transcription in yeast (i.e. —that “autoactivate”)

Summary

Introduction

Yeast two-hybrid systems [1,2,3,4] are standard tools used to identify novel protein-protein interactions and to perform structure-function analysis on previously defined protein-protein interactions. Such systems are effective with a substantial fraction of eukaryotic proteins and have played an important role in high throughput proteomic analyses aimed at establishing sets of interacting proteins All of these potential issues would be expected to be less problematic in the bacterial two-hybrid system. To maximize chances of obtaining all relevant interactors for a protein of interest, it would be desirable to have the capability to rapidly test a given bait in both yeast and bacterial milieus

Methods

Results

Conclusion