PWITCH - A VERSATILE 2-HYBRID ASSAY VECTOR FOR THE PRODUCTION OF EPITOPE ACTIVATION DOMAIN-TAGGED PROTEINS BOTH IN-VITRO AND IN YEAST

We describe the construction of a new vector, pWITCH, designed to faci litate the characterisation of proteins encoded by novel cDNAs isolate d using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while tra nslation of the expressed cDNAs results in proteins which are tagged i n vitro with a specific epitope and in vivo with both the epitope and the VP16 transcription activation domain. The principle of using multi ple promoters each able to operate under different conditions to expre ss different combinations of protein domains without the need for subc loning should be generally applicable.