Study of Molecular Interactions of CCM Proteins by Using a GAL4-Based Yeast Two-Hybrid Screening.

Abstract

The yeast two-hybrid system was originally designed to detect protein-protein interactions using yeast transcriptional activators. Since its original description, this technique has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. Nowadays, the yeast two-hybrid screen remains one of the leading molecular tools to study protein-protein interactions in native intracellular conditions. In these years, the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches.Several groups studying molecular mechanisms underlying the Cerebral Cavernous Malformation disease have successfully used the yeast two-hybrid system or related methods to isolate, identify, and characterize molecular interactions involved in the onset and progression of the pathology.Here we describe general principles, strengths, and limits of the yeast two-hybrid technology, and the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay by using a GAL4-based system.