NanoLuc luciferase as a quantitative yeast two-hybrid reporter

Abstract The yeast two-hybrid (Y2H) assay is a powerful technique to identify protein-protein interactions. However, the auxotrophic markers that are the most common Y2H reporters take several days to yield data and require subjective assessment of semiquantitative data to identify interactions. Several reporters have been developed to overcome these disadvantages, but there is still a need for a Y2H reporter that is objective, fast, and able to be performed with common laboratory equipment. In this report, we replaced the ADE2 reporter in BK100 with NanoLuc luciferase to yield BK100Nano. We developed an optimized assay to measure NanoLuc activity in 96-well plates and analyzed a set of 74 pairs identified in Y2H library screens, which revealed 44 positive interactions using an unbiased cutoff based on the mean luminescence of negative control samples. The same set was also tested for growth on Y2H selection medium via expression of the HIS3 reporter. We found 91% agreement between the two assays, with discrepancies attributed to weak interactions that displayed variable growth on Y2H medium. Overall, the new BK100Nano strain establishes a quantitative and convenient method to identify Y2H interactions and has potential to be applied to a high throughput manner.