A versatile bait vector allowing rapid isolation of prey vectors in Gal4-dependent yeast two-hybrid screens

Two-hybrid screens have been widely used in recent years for identifying an d isolating new interaction partners to known proteins. Current Gal4-depend ent two-hybrid screens employ mostly bait and library vectors, which both h ave the ampicillin gene as a selection marker in bacteria. The isolation of the library plasmids takes several days, because library and bait plasmid cannot be separated easily. We have replaced the ampicillin gene by a kanam ycin gene in a Gal4 DNA binding domain bait vector. This vector reduces fou r- to fivefold the time period required for the isolation of library plasmi d DNA. In addition we have changed the multicloning site in the modified ve ctor for easy cloning of cDNA inserts. This vector is advantageous not only in standard two-hybrid screens, but also in mass screens that require mult iple screening rounds in order to characterize networks of protein-protein interactions. (C) 1999 Academic Press.