Non-canonical Split Intein Based Controllable C-cleavage

Intein-based protein cleavage has been used in protein purification,peptide ligation and cyclization.However,the existing methods employing conventional contiguous inteins often result in spontaneous cleavage and cause reduced yields of the desired protein products.Here we report a controllable cleavage strategy using three engineered S1 split-inteins,Ter ThyX,Ssp GryB and Rma DnaB.In this controllable C-cleavage design,the C-terminus of a S1-IC sequence was fused with thioredoxin to form a precursor protein,and a synthetic S1-IN peptide from the Ssp DnaB S1 split-intein was used together with DTT to trigger the cleavage at the C-terminus of the IC,where this peptide can trigger C-cleavage in all three inteins.This approach provided more options for protein purification using intein C-cleavage without spontaneous cleavage and might be useful to the studies of the intein structurefunction relations.