- ProteinLinks' two-hybrid system utilizes TetR as the DNA binding domain of a bait fusion. TetR is derived from bacterial transposon Tn10, which is evolutionally distant from any yeast proteins. It alleviates endogenous interference from yeast proteins during yeast two-hybrid screening. Therefore, the TetR-based two-hybrid system is much more sensitive than Gal4-based two-hybrid systems.
- ProteinLinks' two-hybrid system has TetOp-URA3 and TetOp-LacZ repoters in two distinct basal promoters. Both the URA3 and LacZ reporters can be quantitatively assayed for the affinity estimation.
- The TetOp-URA3 reporter is more sensitive than that of previously described LacZ, LEU2 and HIS3 reporters , which should therefore facilitate detection of even weaker and perhaps more physiologically relevant interactions.
- Sensitivity of the URA3 reporter can be down-regulated in two ways. Expression of the URA3 reporter can be titrated by 6-azauracil, an inhibitor of the URA3 gene product. The URA3 reporter activity can also be reduced by tetracycline and its derivatives, which disrupt binding of the TetR bait to Tet-Op. Either of the two inhibitors diminishes the sensitivity of the URA3 reporter, allowing the use of Proteinlinks' two-hybrid system with baits that activate transcription.
- The URA3 reporter allows the use of 5-FOA to select against its gene expression. This unique feature allows potential identification of small molecules that disrupt specific protein-protein interactions.
- ProteinLinks' two-hybrid system eliminates common false positives that have plagued with other two-hybrid systems by examining the dependency of reporter gene expression on the TetR-bait/TetOp interaction.