Interaction-dependent native chemical ligation and protein trans-splicing (IDNCL-PTS) for detection and visualization of ligand-protein interactions

摘要

abstract_textpWe constructed a detection system to visualize ligand-protein interactions by specific enzyme activity based on interactiondependent native chemical ligation and protein trans-splicing (IDNCL-PTS). We engineered b-galactosidase (bGal) capable of generating enzyme activity through protein trans-splicing (PTS) using a split intein derived from Synechocystis sp. PCC6803 (Ssp) DnaB. Two short peptide reactive tags, each having the sequence of bGal(24-34) or DnaB(1-11), were incorporated into a ligand and a target protein, and the ligand-protein interaction promoted the ligation between the peptide tags by a proximity effect. Combining IDNCL and PTS, interactions of carbohydrates with maltose binding protein (MBP) and phosphopeptides with peptidyl-prolys cis/trans isomerase NIMA-interacting 1 (Pin1) were clearly detected by bGal activity. Interestingly, phosphopeptide-Pin1 interactions were visualized directly in crude periplasmic extracts./p/abstract_text