Protocol

The Bacterial Two-Hybrid System as a Reporter System for Analyzing Protein-Protein Interactions

This protocol was adapted from "Bacterial Two-Hybrid System for Studying and Modifying Protein-Protein Interactions," Chapter 11, in Protein-Protein Interactions, 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The bacterial two-hybrid system provides a convenient method for rapidly and quantitatively assessing the effect of specific mutations on protein-protein interactions. It is well suited to analyzing series of mutants (e.g., those generated from alanine scanning experiments or comprehensive mutagenesis of DNA-binding sites). The Escherichia coli lacZ gene (encoding ß-galactosidase) serves as a reporter gene because its expression is easily measured using a quantitative assay. This protocol describes methods for assessing the interaction of hypothetical proteins X and Y in the bacterial two-hybrid system. Plasmids expressing the hybrid proteins DBD-X and RNAP-Y are constructed. Protein X can be fused either to a monomeric DBD (the zinc finger domain from the Zif268 protein) or to the dimeric bacteriophage cI repressor protein. Protein Y can be fused either to the monomeric E. coli RNAP -subunit or to the dimeric E. coli RNAP . Plasmids encoding these hybrid proteins are used to transform an appropriate lacZ reporter strain. Quantitative ß-galactosidase assays are performed to measure the effect of the two-hybrid proteins on reporter gene expression. Following successful demonstration of X-Y interaction in this system, these protocols can be used to assess the effects of mutations on the interaction of X and Y.