pBridgeTM expresses two proteins: a DNA-binding domain fusion, and an additional protein (1–3). pBridge thus allows establishment of three-hybrid systems when used in combination with an activation domain fusion vector and yeast strains from any of CLONTECH's GAL4-based two-hybrid systems (#K1604-1, #K1605-1, #K1612-1). This vector generates a hybrid protein that contains the sequences for the GAL4 DNA-binding domain (DNA-BD; a.a. 1–147) and the sequence cloned into MCS I. The fusion protein is expressed in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (NLS) that are an intrinsic part of the GAL4 DNA-BD (3). An additional gene of interest can be cloned into MCS II which is located downstream of an HA epitope and a second NLS. The resulting fusion protein is conditionally expressed from the MET25 promoter in response to methionine levels in the medium; i.e., it is repressed in the presence of 1 mM methionine and expressed in the absence of methionine (1).

         pBridge is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae. It carries the bla gene (for ampicillin resistance in E. coli) and the TRP1 nutritional marker that allow yeast auxotrophs carrying pBridge to grow on limiting synthetic medium lacking tryptophan. Note: Yeast strain Y187 is a methionine auxotroph; therefore, haploid Y187 harboring pBridge cannot be grown on medium lacking methionine.