( vity is restored. Therefore by creatin...)New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

vity is restored. Therefore, by creating genetic fusions between the gene of interest to one domain and its interacting partner to the other domain, interaction of the two proteins results in restoration of the activation of the transcriptional activator. Genetic selection for this interaction is provided by an amino acid biosynthetic enzyme that is absent in the yeast host and whose expression becomes induced only when the transcription activator is reconstituted. This two-hybrid system has been widely used.

Despite these successes, the Fields yeast two-hybrid system does not represent a universal system for gene discovery by protein-protein interactions. There are significant limitations to the Fields method that make research into developing experimental alternatives to this procedure a high priority. A potentially significant limitation is the host organism, yeast, that is used for identifying protein-protein interactions. Although the yeast is a eukaryote, it is far removed from human, mammalian, and higher eukaryotic organisms. For example, yeast does not carry out some of the posttranslational modifications (e.g., most tyrosine-phosphorylation events) that are responsible for important protein interactions in mammalian cells.^5,6 In addition, the environment of a yeast cell is so different from higher eukaryotes that uncertainties about the proper folding of these proteins are valid.⁷ Finally, a number of proteins of interest have been discovered to be toxic when expressed in yeast.⁷ This further underscores the limitations of using a single host organism to study eukaryotic proteins. Clearly, developing a system to study protein-protein interactions using a mammalian host organism would obviate many of these concerns and provide an attractive method for uncovering the biological role of many gene products that cannot be studied in the yeast. The mammalian two-hybrid syst
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