A powerful method to detect protein interactions in mammalian cells
Tanya Hosfield Cathy Chang
With Stratagenes mammalian two-hybrid assay kit, hybrid genes are used to detect protein-protein interactions via the activation of reporter gene expression. This expression occurs as a result of reconstitution of a functional transcription factor caused by the association of two hybrid proteins. It allows researchers to study interactions in vivo between mammalian proteins that require posttranslational modification or external stimulation not present in lower eukaryotes.
Protein-protein interactions in mammalian cells are the basis of virtually every cellular process, such as DNA replication, transcription, translation, splicing, secretion, cell cycle control, signal transductions, and intermediate metabolism.1 As efforts to sequence whole genomes result in data for vast numbers of new proteins, there is a corresponding need to identify interactions among these proteins. Methods used to discover these interactions will be extremely helpful in elucidating the in vivo function of newly discovered genes and, in turn, will aid in further understanding known protein functions.
One of the most powerful methods for studying protein-protein interactions is
the yeast two-hybrid system developed by Dr. Stanley Fields and colleagues.2
This system has been highly useful for detecting and identifying protein-protein
interactions in vivo. The Fields two-hybrid system, and the many
second-generation versions currently used,2,3,4,5 exploit the modular
nature of a transcriptional activator. Transcriptional activators, such as the
GAL4 protein of yeast, contain a DNA binding domain and an activation domain
that can be separated. If these domains are then reconstituted in trans,