Optimization of Neh2-Luc Reporter for Screening of Activators of Antioxidant Program.

Reporter vectors expressing of amino acid residues 16–96 and 16–85 from Neh2-domain of Nrf2 transcription factor fused to firefly luciferase have been constructed. Cell lines stably expressing the above reporter constructs as well as an earlier developed Neh2-luc fusion reporter have been generated using HeLa cells. Comparative analysis of all three reporter lines has been performed using a well-known Nrf2 activator, nordihydroguaiaretic acid. The reporter expressing the minimal fragment of Neh2-domain containing 16–85 aa sequence exhibits a 2-fold higher amplitude of activation compared to Neh2-luc reporter, and thus can be recommended for high throughput screening for Nrf2 activators. A likely reason for the improved response of the above reporter could be ubiquitination of N-terminal lysine residues (7, 10, 11) of luciferase protein adjacent to the ubiquitinated Neh2 sequence. [ABSTRACT FROM AUTHOR]