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

Abstract

AbstractWe constructed a detection system to visualize ligand‐protein interactions by specific enzyme activity based on interaction‐dependent native chemical ligation and protein trans‐splicing (IDNCL‐PTS). We engineered β‐galactosidase (βGal) 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 βGal(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 βGal activity. Interestingly, phosphopeptide‐Pin1 interactions were visualized directly in crude periplasmic extracts.