Single‐step affinity purification of recombinant proteins using a self‐excising module from Neisseria meningitidis FrpC

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self‐excising module allowing single‐step affinity chromatography purification of untagged recombinant proteins. It consists of a 250‐residue‐long self‐processing module of the Neisseria meningitidis FrpC protein with a C‐terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site‐specific cleavage of the Asp–Pro bond linking the target protein to the self‐excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self‐excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione‐S‐transferase, maltose‐binding protein, β‐galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin‐binding domain or poly‐His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self‐splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.