Split inteins are proteins that self-catalyze protein trans-splicing (PTS) and widely used in biotechnology. In our laboratory, we developed an engineered Npu DnaE 304-2 intein by changing the split position to 11 residues of N-intein and 126 residues of C-intein and substituting 5 amino acids to improve the PTS activity. Although the PTS activity is greatly reduced when the split position was altered to 131-residue N-intein and 6-residue C-intein, by fusing the interacting peptide and protein to each of the N-inteins and C-inteins, a system was constructed in which the PTS reaction proceeds depending on the peptide-protein interaction. In this case, a split of aminoglycoside 3'-phosphotransferase (APH), an enzyme that inactivates kanamycin, was used for the extein, and active enzyme is generated upon PTS reaction. In the present study, we have detected the interaction between the peptides and murine double minute 2 (MDM2) using the engineered split intein and APH, and constructed the selection method to find the peptides that bind to MDM2.

We designed the protein bearing N-APH, N-intein, and MDM2. A MDM2-binding peptide, PMI (TSFAEYWNLLSP), was used as a model. We designed the peptide bearing PMI, 6-residue C-intein, and 7-residue C-APH. We also designed the alanine variant of PMI to compare the binding affinity with the PTS activity. When PMI or its alanine mutant interacts with MDM2 inside E. coli, it is expected that PTS occurs to generate active APH. The protein and the peptides were expressed in E. coli and cultured on LB-agar plate containing kanamycin. When the peptides having higher binding affinity to MDM2 (Kd < 1 µM) were used, E. coli cells were grown in the presence of kanamycin. We have also attempted to discover the peptides that bind to MDM2 using a random peptide library.