A sensitive, semi-quantitative mammalian two-hybrid assay

Abstract

Protein–protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system. This inducibility, combined with additional improvements of the iM2H components, results in an up to 100-fold increase in sensitivity compared with conventional M2H approaches. In addition, we include a number of reference interactions in our iM2H approach, which enable semiquantitative assessment of protein interactions. Using Groucho/Tle proteins and their binding partners, we demonstrate the applicability of our iM2H to established protein networks. Finally, to test the applicability of our system for drug screening, the interference of a small-molecule inhibitor on a known protein–protein interaction was tested, and the particular advantages of the internal reference interactions were shown.

Keywords

• protein–protein interactions
• mammalian two-hybrid