Modification of C‐segment of Cfa DnaE split intein for improving clean‐in‐place in chromatography process

This research focuses on the construction of an affinity purification system based on Cfa DnaE split intein. Cfa DnaE intein is an artificially constructed intein with the advantages of a fast cleavage reaction and good stability. In a previous study, a purification system that uses Cfa intein as a tag was constructed, the separation of the target protein and the tag during the purification process was completed, and the purity of the purified target protein reached 98.21%. Guided by molecular docking results, we identified flexible regions in the split intein and inserted several glycines into the protein to decrease the stability of the Cfa IC, thereby improving the regenerability of the IN media. Inserting 6 glycines between amino acids 14 and 15 of IC improved the regeneration rate of IC‐GFP on the column to approximately 96%.