- ProteinLinks' two-hybrid system utilizes TetR as DNA binding domain of the 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.