Kelch-like ECH-associated protein (Keap1) is implicated in sequestration and ubiquitination of the nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is a transcriptional regulator that promotes the expression of genes that a cytoprotective function in response to oxidative stress. Disruption of the interaction between Nrf2 and Keap1 has been upregulating the expression of cytoprotective oxidative stress response enzymes. On the other hand, we have attempted to develop a selection method to discover peptides that bind to target proteins based on split intein, which self-catalyzes protein trans-splicing (PTS). In the previous study, we engineered Npu DnaE intein. Using an active variant, 304-2 split intein, we altered the split position to reduce the PTS activity. When a ligand and its interaction partner are appended to the N-intein and C-intein, the ligand-protein interaction can promote the PTS reaction. Using an aminoglycoside 3’-phosphotransferase type I (APH) as the splicing product, progress of PTS provides resistance against kanamycin in E. coli. In the present study, we attempted to detect the interaction between peptides and Keap1 by using the split-intein based method. Moreover, we attempted to select the peptides that bind to Keap1.

A peptide derived from Nrf2 was used as the model to detect the peptide-Keap1 interaction. The peptide was conjugated with the C-intein and C-APH. Kelch domain of Keap1 (321-609) was appended to the N-intein and N-APH. We also constructed a circular permutant of Keap1, because the Nrf2-binding surface of Keap1 is far from the N-intein. Both peptides and proteins were expressed in E. coli, and the cells were grown with several concentrations of kanamycin in the presence of isopropyl-ß-D-thiogalactoside (IPTG) on LB-agar plates.