Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system

The yeast two-hybrid system is frequently used to identify protein-protein interactions. The assay is based on the functional reconstitution of a tran scriptional activator. Since an indirect phenotype of the positive clones i s the basis for selection of positive interacting clones, the two-hybrid sc reens are vulnerable to false positives. Here we report a screening protoco l based on the sequential use of the cotransformation approach followed by the genetic method for verifying true two-hybrid interactions, Using this p rocedure, we have screened a cDNA library and have been able to isolate tru e positives from the yeast two-hybrid screen. (C) 2000 Academic Press.